A series of Ca2+ oscillations during mammalian fertilization is necessary and sufficient to stimulate meiotic resumption and pronuclear formation. It is not known how effectively development continues in the absence of the initial Ca2+ signal. We have triggered parthenogenetic egg activation with cycloheximide that causes no Ca2+ increase, with ethanol that causes a single large Ca2+ increase, or with Sr2+ that causes Ca2+ oscillations. Eggs were co-treated with cytochalasin D to make them diploid and they formed pronuclei and two-cell embryos at high rates with each activation treatment. However, far fewer of the embryos that were activated by cycloheximide reached the blastocyst stage compared to those activated by Sr2+ or ethanol. Any cycloheximide-activated embryos that reached the blastocyst stage had a smaller inner cell mass number and a greater rate of apoptosis than Sr2+ -activated embryos. The poor development of cycloheximide-activated embryos was due to the lack of Ca2+ increase because they developed to blastocyst stages at high rates when co-treated with Sr2+ or ethanol. Embryos activated by either Sr2+ or cycloheximide showed similar signs of initial embryonic genome activation (EGA) when measured using a reporter gene. However, microarray analysis of gene expression at the eight-cell stage showed that activation by Sr2+ leads to a distinct pattern of gene expression from that seen with embryos activated by cycloheximide. These data suggest that activation of mouse eggs in the absence of a Ca2+ signal does not affect initial parthenogenetic events, but can influence later gene expression and development.