4-hydroxy-N-methylproline and 4-hydroxy-N,N'-dimethylproline have been identified in the aqueous extracts of Melaleuca spp. Characterization of the two compounds is discussed.Whilst using *H NMR to measure the accumulation of glycinebetaine in a range of native South Australia flora [l] it was observed that a specimen of Melaleuca lanceolata contained a high concentration of an N-methyl hydroxyproline derivative. Subsequent studies have shown that a number of species belonging to the genus Melaleuca accumulated this compound and another hydroxyproline analogue. The levels of these compounds in plant tissue respond to environmental stress (Naidu, B. P., Jones, G. P., Paleg, L. G. and Poljakoff-Mayber, A., unpublished data) suggesting that they may be important in determining the drought and/or salinity resistance of these species. Furthermore, the occurrence of these compounds together with proline and N-methylproline, at different levels in different Melaleuca species holds promise for their use in chemotaxonomic studies. The compounds were isolated by ion-exchange chromatography and identified as ( - )-(2S, 4R)-1-methyl-4-hydroxypyrrolidine-2carboxylic acid (4-hydroxy-N-methylproline, 1) and (-)-(2S, 4R)-1,1'-dimethyl-4-hydroxypyrrolidine-2-carboxylic acid (4-hydroxy-N,N' dimethylproline, betonicine, 2) by 'H and 13C NMR and by comparison with authentic compounds. X-ray crystallographic analyses of the two compounds enabled unambiguous confirmation of their identities. The conformations of 1 and 2 determined by X-ray diffraction studies of their respective hydrochlorides are shown below. Oxygen and nitrogen atoms are lightly and heavily shaded respectively. The orientation of the N-methyl group with respect to the carboxyl group in 1 is the same as that predicted by an earlier 'H NMR study [5].1 and 2 have both been found in higher plants [2-4] and algae [5-7] at levels less than 0.5 % dry weight. In the present case levels of 1 of >4 % dry weight were measured in M. lanceolata and > 1.4 % dry weight of both 1 and 2 in leaves of M. uncinata. 'H and 13C NMR spectral data for 1 are consistent with that reported previously [5]. The *H NMR spectrum of 2, as the hydrochloride (90 MHz, D20, pH l.O), closely resembled that of 1 and showed peaks at δ3.19 (3H, N-Me) and δ3.47 (3H, N-Me). A complex multiplet at δ2.25-3.00 (2H) was attributed to H-3a and H-3b. H-5a and H-5b each gave a doublet of doublets centred at δ3.62 and δ4.11 respectively as part of an AMX pattern involving H-4 where J5a,5b = 13.0 Hz, J5a,4 = 4.4 Hz and J5b,4 = 6.0 Hz. A multiplet at approximately δ4.7, which was significantly obscured by the solvent peak, was ascribed to H-2 and H-4. When the pH was adjusted to 4, a part of this multiplet moved upfield to form a broad doublet at δ4.36 (1 H), J = 11 Hz which was ascribed to H-2.The 13C NMR spectrum of 2 (22.5 MHz, D20, pH 1.0) shows a singlet at δ 171.0 (-COOH), two doublets at δ77.5 and δ76.8 (C-2, C-4), two triplets at δ69.1 (C-5) and δ38.1 (C-3) and two quartets at δ57.6 and δ52.0 (N-Me, N-Me). At pH 4.0 the carboxyl signal moved downfield to δ 173.0 and the signals associated with C-2 and C4 merged to form a single doublet at δ76.9.It is noted that the melting points of the hydrochlorides for both 1 and 2 are significantly lower than those reported previously [2,5] and this is attributed to the formation of different crystalline modifications.