In this study, the activity of mouse phospho-glycerate kinase promoter (PGK) and murine embryonic stem cell virus promoter (MESV), enhanced by the connection of the R-segment and part of the U5 sequence (RU5) of the long terminal repeat of human T-cell leukemia virus type 1, was compared with that of simian virus 40 early promoter (SV40) and human cytomegalovirus early promoter (CMV). Escherichia coli β-galactosidase (LacZ) reporter gene connected to these promoters was microinjected into pronuclei of mouse zygotes, and their expression of developing embryos during the preimplantation period was evaluated histochemically with X-gal. No difference was observed in the proportion of embryos which developed into the morula stage among the promoter sequences at 96 h after hCG injection, but the expression of LacZ gene connected to MESV-RU5 (MESV-LacZ) was lower than that to PGK-RU5 (PGK-LacZ), CMV (CMV-LacZ) and SV40 (SV40-LacZ) in the morula stage embryos (P<0.05). In another experiment, more than 50% of embryos microinjected with PGK- and CMV-LacZ responded positively to X-gal staining at 48 h after hCG injection and the activity of these promoters continued at nearly the same rate from there onwards. However, the rate of expression of SV40- and MESV-LacZ was lower than that of PGK- and CMV-LacZ at 48 h after hCG injection (P<0.05). Although expression of MESV-LacZ was consistently low in proportion and weak in intensity, that of SV40-LacZ was high at 72 h after hCG injection and was equivalent to that of PGK- and CMV-LacZ at 96 h after hCG injection. Regardless of the promoters used, the expression of LacZ gene in the embryos showed various intensities of blue staining. The frequency of mosaic patterns and a weak intensity of gene expression in morphologically normal embryos had a tendency to be higher than in degenerated embryos or those whose development had been arrested.