Multiparameter flow cytometry (FCM) measurements were made on acridine orange (AO)-stained mouse testicular cells and epididymal sperm cells to determine the effects of varying dosages of thiotepa (0-5 mg/kg ip daily X 5 days) on spermatogenesis at 7, 28, and 67 days after the last exposure (ALE). FCM multiparameter measurements included DNA stainability vs RNA content, peak amplitude vs integrated area of DNA fluorescent signal, and double-stranded DNA vs single-stranded DNA. Thiotepa exhibited dramatic damaging effects on the kinetics and/or cell kill of seven testicular cell types measured by dual-parameter flow cytometry. At 7 days ALE, one 4N cell type, likely the pachytene spermatocyte, was absent from the testes, and another was reduced by about 70%. By 28 days ALE, most of the germ cells were absent from the seminiferous tubules, and by 67 days ALE the testes were undergoing recovery of spermatogenesis with only half of the seminiferous tubules repopulated after treatment with 5.0 mg/kg. The dual parameters of DNA stainability vs RNA content provided better resolution of testicular cell types into distinct populations than the peak vs area processing of the green fluorescent signal of AO-stained cells. Dosage of thiotepa was significantly related to percentage of sperm head morphological abnormalities assayed by light microscopy. Utilizing the metachromatic properties of acridine orange, FCM measurements of the amount of single-stranded DNA induced within acid-stressed whole sperm or heat-stressed nuclei detected alterations of chromatin structure at the same minimal effective dose required to increase abnormal sperm head morphology. Epididymal sperm isolated from mice exposed to some concentrations of thiotepa had an increased percentage of free heads and tails. DNA in free heads denatured in situ to a greater extent than DNA in intact sperm.