N,N',N''-triethylene thiophosphoramide (Thio-TEPA) is an alkylating agent whose antineoplastic activity has been known for nearly 30 years. Human plasma pharmacokinetic studies revealed the presence of TEPA, a Thio-TEPA metabolite which after 4 h achieved plasma concentrations equal to those of the parent compound. We studied the activity of both Thio-TEPA and TEPA against murine leukemia P388 cells in culture. We found that Thio-TEPA is approximately two-fold more active than TEPA in arresting cell growth (IC50 = 2.8 microM for TEPA and 1.5 microM for Thio-TEPA). In inhibiting [3H]thymidine incorporation, Thio-TEPA and TEPA have the same activity (IC50 = 2 microM for both compounds). Experiments in which drug was removed from cell cultures which were further incubated in drug-free media, revealed that the bulk of the cell damage occurs during the first 4 h of incubation. Cell cultures exposed to 0.5 microM Thio-TEPA for 22 h fully recovered their [3H]thymidine incorporation ability after 24 h of drug-free incubation. Cells exposed to 2.5 microM Thio-TEPA for 22 h partially recovered their ability to incorporate [3H]thymidine. Cells exposed to 10 microM Thio-TEPA for 22 h did not recover their ability to incorporate [3H]thymidine. Gas liquid chromatographic analysis of the media from incubated cells showed that the concentration of Thio-TEPA remained unchanged during the incubations and that TEPA was not present. In Thio-TEPA doses ranging from 0.1 microM to 100 microM, [3H]uridine and [3H]-leucine incorporation were less affected than [3H]thymidine incorporation. This may indicate that a longer observation time may be needed to allow the DNA damage to be expressed in terms of protein or RNA synthesis.