After fertilization in the mouse, the zygotic genome is activated in two-cell embryos by the spontaneous expression, among other genes, of the major inducible heat shock gene, hsp68, in the absence of heat-inducibility of heat shock genes. To obtain information on this phenomenon, we have probed one- and two-cell embryo's ability to express microinjected reporter DNA constructs, containing the Escherichia coli lacZ gene driven by promoters from early SV40 genes, the human beta-actin gene, and the normal or HSE-deleted mouse hsp68 gene. Activity of these promoters was also tested in mouse granulosa cells and dictyate oocytes, as a function of circular/linear construct configuration and occurrence of heat shock. The hsp68 promoter was heat-inducible in both granulosa cells and oocytes. Its heat activation required the presence of HSEs and, in the oocytes, of construct linear configuration. In the embryos however, this promoter was expressed independently of the presence of HSEs and of construct configuration, and its activity was not affected by heat shock. When constructs with early SV40 and beta-actin promoters were injected into one-cell embryos, they appeared to be inactivated with the first embryonic cleavage, in agreement with previous observations [Wiekowski et al., 1992]. By contrast, both normal and HSE-deleted hsp68 promoters maintained their activity through the first cleavage, providing the first evidence of a gene escaping such transcriptional repression. Present results confirm previous findings on hsp68 expression during early mouse development, and suggest that this activation is mediated by a factor(s) other than HSF.