The induction and nature of mutations in the lacI transgene were evaluated in multiple tissues after exposure of adult male B6C3F1 lacI transgenic mice to cyclophosphamide (CP). Mice were given a single i.p. injection of 25 mg CP/kg, 100 mg CP/kg, or vehicle (PBS) and then necropsied 6 weeks after treatment to allow DNA extraction and lacI mutant recovery. Tissues evaluated included target tissues for tumorigenesis (lung, urinary bladder) and sites not susceptible to tumor formation in B6C3F1 mice (kidney, bone marrow, splenic T-lymphocytes). After exposure to the high dose of CP, a significant increase in the mutant frequency (Mf) was detected in the lungs and urinary bladders, compared to the respective tissues from vehicle-treated controls. In contrast, the Mfs in kidney, bone marrow, and splenic T cells from CP-treated mice were not significantly different from controls. The spectra of mutations in lacI from lung and urinary bladder were significantly changed after high-dose CP treatment, with a significant increase in the frequency of A. T --> T. A transversions found in both tissues and a significantly elevated frequency of deletions in the lungs. Conversely, in vehicle-treated mice, the two predominant classes of lacI mutations recovered in lung and urinary bladder were G. C --> A. T transitions at CpG sites and G. C --> T. A transversions. These CP exposures were also genotoxic as measured by the significant induction of micronuclei in peripheral blood 48 hr after exposure. These data indicate that under these study conditions, CP-induced mutations are detectable in the lacI transgene in the target tissues, but not in nontarget tissues for CP-induced cancer. With the lacI assay it is possible to study mutagenicity in a variety of critical tissues to provide mechanistic information related to genotoxicity and carcinogenicity in vivo.