A convenient route is described for attachment of acyl groups CO(CH2)nN(Et)2(CH2)mNH(Et)2 (n = 3, m = 2; n = 4, m = 2-4), CO(CH2)nN(Et)2(CH2)mNEt3 (n = 4, m = 2-4), or CO(CH2)4N(CH2CH2)3N(CH2)nCH3 (n = 1 or 9) to O-3' of thymidine 5'-phosphate (TMP). The compounds are prototypes of 5'-nucleotide derivatives in which the two anionic charges could become partially masked in intramolecular anionic-cationic interactions and which might be able to diffuse into mammalian cells to furnish intracellular antimetabolite 5'-nucleotides by hydrolytic loss of a dicationic 3'-O-acyl group. At pH 7.6, 37 degrees C, hydrolyses of the 3'-ester linkages were pseudo first order with t1/2 values in the range 28-85 h. Paper chromatography in n-PrOH-H2O at pH 7.6 showed that type 1 or 2 derivatives were equally or slightly less hydrophobic than TMP (Rf 0.24), whereas the n-decyl type 3 compound (Rf 0.66) was markedly more hydrophobic, apparently because chain branching in group 3 is less than in 1 or 2. A sensitive and specific assay was developed for liberation of intracellular TMP in cultured mouse L fibroblasts in which synthesis of TMP and, hence, of DNA was suppressed by a combination of aminopterin and 5'-amino-5'-deoxythymidine (5'-NH2-dT). The TMP derivatives (100 microM) stimulated DNA synthesis, but omission of 5'-NH2-dT increased stimulation 6.5-fold, suggesting that stimulation occurred via degradation of the derivatives to dT. In confirmation, derivatives of type 2 (n = 4, m = 2 or 4) (100 microM) or type 3 (n = 9) (200 microM), in the presence of aminopterin, did not stimulate DNA synthesis in the LM(TK-) strain of L cells, which is genetically deficient in dT kinase. TMP (1 mM) stimulated DNA synthesis 2-3-fold and appeared to enter LM(TK-) cells without dephosphorylation, because dT (1 mM) gave no stimulation. If TMP is assumed to enter solely by passive diffusion, the inactivity of the TMP derivatives can be ascribed in part to their 2-fold higher molecular weight which can be expected to reduce flux through natural membranes ca. 16-fold.