In transgenic animals, genotype-specific modifiers exert a control over transgene methylation and expression that may or may not be position dependent. These factors belong to different classes, some of them possibly related to modifiers of position-effect variegation in Drosophila. The study of hepatitis B virus (HBV) gene expression in transgenic mice has revealed the existence of many factors influencing transcription, including hormones and tissue-specific transcription factors. We now report the effect of genotype-specific modifiers on HBV surface antigen (HBsAg) expression and transgene methylation. Compared with the C57BL/6 background, the DBA/2 and 129sv backgrounds cause enhancement of HBsAg expression, with little or not effect on transgene methylation or transcription. In contrast, a single cross with a BALB/c mouse is responsible for de novo methylation and silencing of the transgene in all offspring. Several modifiers appear to segregate in the progeny of a transgenic E36 male mouse crossed with (C57BL/6 x BALB/c) F₁ females, with the emergence of a high-expressor group. Our observations suggest that different modifiers act cooperatively, at both the transcriptional and post-transcriptional levels, as part of a complex system regulating transgene expression. This transgenic model provides a system to genetically map new mouse strain-specific modifiers, some of them involved in epigenetic modification and transcription control.