The synthesis and biological activity of four novel analogues of the cytostatic and antimitogenic agents chlamydocin and HC-toxin are reported in which the natural products' reactive epoxy ketone side-chain moiety is replaced by a chloromethyl or a diazomethyl ketone functionality, but the respective 12-membered cyclic tetrapeptide ring systems are retained. Syntheses of the linear tetrapeptide sequences were, in each case, achieved by conventional methodology and designed such that cyclization would be onto proline. The use of suitably protected L-2-aminosuberic acid (Asu) enabled the ready assimilation of the desired chloromethyl and diazomethyl ketone functionalities after cyclization. Cyclization was accomplished by using bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl). Yields of cyclic product were comparable to or, in the case of the HC-toxin ring system, better than those previously reported. Liberation of the Asu-side-chain acid and manipulation to the required functionalities via mixed anhydride to the diazomethyl ketone and quenching with HCl to yield the chloromethyl ketone was achieved in excellent yield for the HC-toxin analogues but in only moderate yield for the chlamydocin analogue. The antimitogenic activities of HC-toxin chloromethyl ketone (IC50 = 30-40 ng/mL) and chlamydocin chloromethyl ketone (IC50 = 3-10 ng/mL) were found to be 3-4-fold lower than those of the natural products themselves. The diazomethyl ketone analogue of HC-toxin was found to be inactive (IC50 greater than 2000 ng/mL). A modification of the HC-toxin peptide ring system, [L-Phe]3-HC-toxin chloromethyl ketone was found not to be a more active analogue (IC50 = 40-100 ng/mL). The nature of the putative target molecule, the binding interactions of the various analogues and the contribution of rate of inhibition toward activity are briefly discussed. The chloromethyl ketones herein reported constitute the most potent synthetic antimitogenic cyclic tetrapeptide analogues yet designed.