Using flow cytometry and conventional spectrofluorimetry we have previously shown that chloroethylnitrosoureas (CNUs) can exhibit marked inhibition of cellular enzymes catalysing hydrolysis of fluorescein diacetate (FDA). More potent inhibition was seen for the carbamoylating CNUs, whereas alkylating agents were largely inactive. We now report results obtained with the developmental imidazotetrazines mitozolomide and temozolomide in comparison with BCNU, the novel alkylating agents clomesome and cyclodisone, and the active mitozolomide metabonate MCTIC. Inhibition of EMT6 mouse mammary-tumour esterases was seen for mitozolomide and temozolomide, and activity against purified porcine carboxylesterase was demonstrated. Flow cytometric analysis showed that inhibition occurred across the entire EMT6 cell population, with no evidence of a subpopulation resistant to enzyme inhibition. Inhibitory potency for the imidazotetrazines was much weaker than for BCNU. With EMT6 cells, I50 values from flow cytometry were 9.7 x 10(-3) M and 1.5 x 10(-3) M for mitozolomide and temozolomide compared with 3.7 x 10(-4) M for BCNU. These were higher than the ID50 values for in vitro antitumour activity (MTT assay), 8.5 x 10(-6) M in the case of mitozolomide and 1.2 x 10(-5) M for BCNU, but similar to that of 5.6 x 10(-4) M for the less toxic temozolomide. MCTIC and cyclodisone showed very low activity, but significant inhibition was seen for clomesome. The results are consistent with the view that the imidazotetrazines do not exhibit major carbamoylating ability, although significant effects are seen at cytotoxic concentrations of temozolomide. In addition, the potential for the generation of carbamoylating species at the enzyme active site cannot be ruled out.