Germ cell mutagenesis was studied in male λlacZ transgenic mice in such a way that the data can be compared with literature data for germ cell mutagenesis obtained with the specific-locus test. This comparison is of interest for validation of the transgenic mouse model. We studied mutagenesis induced by ethylnitrosourea (ENU) in mature spermatozoa isolated from epididymis and vas deferens. In order to investigate mutagenesis in different phases of spermatogenesis, animals were killed at various time points after treatment. After an ENU dose of 150 mg/kg, an increase of the mutant frequency (MF) occurred only in stem cells (9-fold; 100 days post-treatment) but not in post-stem cells (0.1 and 7 days post-treatment). Specific locus test data from literature showed a larger induction of mutations in stem cells (44-fold; > 42 days post-treatment). Although spermatogenesis in mice normally takes ~42 days, we found only a limited increase of the MF at 50 days post-treatment. This may be due to a delay of spermatogenesis, which was confirmed by the observation that O6-ethylguanine levels in spermatozoan DNA remained approximately the same between 0.1 and 50 days and were reduced to background levels at 100 days. Dose-response studies with the 3ilacZ mice indicated a threshold for mutation induction in stem cells at low ENU dosages, which is in accordance with the specific-locus test data. In summary, our experiments suggest that 1lacZ transgenic mice are a suitable model to study germ cell mutagenesis in spermatogonial stem cells.