Double minutes (DM) were found to be present in six of seven clones derived from a 16-day female Mus musculus X M. caroli fetus. The DM-positive clones derived from three primary populations independently set up from the fetus, and included clones with an active M. caroli X chromosome as well as clones with an active M. musculus X. The simplest explanation of these findings is that DM were already present in cells of the M. musculus X M. caroli embryo at the time of X chromosome inactivation and persisted during in vivo development and in vitro culture. This suggests that gene amplification occurred in the early embryo, or even the fertilized egg, perhaps because of interactions between components of germ cells contributed by the M. musculus and M. caroli parents. Alternatively, induction may have occurred independently in these lines, requiring that amplification is an unusually common occurrence in cells from interspecific hybrids.