Mitogen-activated protein kinase (MAPK) pathways mediate some important cellular processes and are likely to also regulate preimplantation development. The role of p38 MAP kinase signaling during murine preimplantation development was investigated in the present study. p38 MAPK, p38-regulated or -activated kinase (PRAK; MK5), map kinase-activated protein kinase 2 (MK2), and heat shock protein 25 (hsp25) mRNAs and proteins were detected throughout preimplantation development. Two-cell stage embryos cultured in the presence of SB220025 and SB203580 (specific inhibitors of p38 MAPK alpha/beta), progressed to the eight-cell stage with the same frequency as controls; however, treated embryos halted their development at the 8- to 16-cell stage. In addition, embryos treated with p38 MAPK inhibitors displayed a complete loss of MK2 and hsp25 phosphorylation and also a complete loss of filamentous actin as indicated by the absence of rhodamine-phalloidin staining. In these inhibitor-treated groups, the embryos were composed of a mixture of compacting and noncompacting cells, and the embryos were one to two cell divisions behind controls. Treated embryos remained viable as the developmental blockade was rescued by removing embryos from the drug treatment and placing them in drug-free medium until they progressed to the blastocyst stage. This study demonstrates that p38 MAPK activity is required to support development through the murine preimplantation interval.