Transgenic mice expressing a tamoxifen-inducible Cre recombinase specifically in cardiomyocytes were generated in 2001 and are in widespread use, having been employed in >150 published studies. However, several groups recently have reported that tamoxifen administration to these mice can have off-target effects that include cardiac dysfunction, fibrosis, and death. For this reason, among others, we considered it important to better characterize the transgene (termed herein, CM-MCM) and its chromosomal location(s). Cytogenetic analysis positioned the CM-MCM transgene within the C band of chromosome 19, and more precise mapping, using genome walking and DNA sequencing, showed that transgene insertion is in the C1 region. Using the genome walking data, we have developed PCR assays that not only identify mice that carry the transgene, but also distinguish homozygous animals (CM-MCM(Tg/Tg)) from hemizygous (CM-MCM(Tg/0)), permitting the rapid assessment of transgene zygosity and, thereby, helping to minimize off-target tamoxifen-induced effects. Substantial rearrangement/duplication of transgene elements is present, and transgene integration was accompanied by the deletion of a 19,500 bp fragment of genomic DNA that contains the promoter, exon 1 and part of intron 1 of the APOBEC1 complementation factor (A1cf) gene, as well as several elements that are predicted to regulate chromosomal architecture. A1cf protein expression is ablated by the deletion and, therefore, homozygous mice are functionally A1cf knockout. The implications of this unexpected finding are discussed.