Strain-specific modifier of DNA methylation 1b (Ssm1b) is a Kruppel-associated box (KRAB) zinc finger gene that promotes CpG methylation in the mouse transgene HRD (Heavy chain enhancer, Rearrangement by Deletion). Ssm1b expression and concomitant HRD methylation are also present in the male and female germ cells of adult mice. Ssm1b is expressed in both diploid (2N) and haploid (1N) oocytes, as well as in 1N spermatids and spermatozoa, but not in 2N spermatogonia. Interestingly, Ssm1b mRNA is not detected in any other adult mouse organ examined, although Ssm1-family mRNAs are highly expressed in the heart. Reflecting strain specificity, Ssm1b expression and HRD methylation are not observed in early-stage C3H/HeJ mouse embryos; however, an Ssm1b-like gene that closely resembles an Ssm1b-like gene previously found in wild-derived mice is expressed in cultured embryonic stem cells derived from C3H/HeJ embryos, suggesting that culture conditions affect its expression. Collectively, this work demonstrates that HRD methylation by Ssm1b is more temporally restricted during spermatogenesis compared to oogenesis, and is altered when embryonic stem cells are cultured from C3H/HeJ inner cell mass cells. This article is protected by copyright. All rights reserved.