Non-radioactive in situ hybridization with mouse centromere specific (major) gamma satellite DNA probe was used to analyze the mechanism of induction of spermatid micronuclei (MN) caused by the alkylating agent mitomycin C (MMC), the spindle poison vinblastine sulfate (VBL) or the DNA topoisomerase II inhibitor etoposide (VP-16). Male mice were treated with a single i.p. injection of 25 mg/kg VP-16, 5 mg/kg MMC or 2 mg/kg VBL, respectively. After 24 h (VP-16, VBL) or 13 days (MMC) stage I spermatid slides were prepared and in situ hybridization was performed using a polymerase chain reaction amplified mouse (major) gamma satellite DNA probe. The observed MN frequencies for VP-16 and MMC, 6.2/1000 and 7.5/1000 round spermatids, respectively, show a strong mutagenic effect on mouse germ cells compared with controls (1.4/1000 spermatids). VBL, on the contrary, induced a much lower total frequency of MN (2.8/1000 spermatids) compared with previous results on mouse somatic cells. Of MN in controls, 24% carried a FISH signal. After correcting for background, MMC induced 38.6% signal-positive MN, consistent with a predominantly clastogenic mode of action, while VBL induced 67.9% signal-positive MN, consistent with a mainly aneugenic mechanism. VP-16 induced 65.5% signal-positive MN, indicating that its MN-inducing capacity is mainly due to whole chromosome lagging.