p38 MAPK isoforms are important in the regulation of a variety of cellular processes. Among the four described p38 isoforms, p38 alpha, beta, and delta are expressed in keratinocytes (Dashti, S. R., Efimova, T., and Eckert, R. L. (2001) J. Biol. Chem. 276, 8059-8063). However, very little is known about how individual p38 isoforms regulate keratinocyte function. In the present study, we use okadaic acid (OA) as a tool to study the role of p38 MAPKs as regulators of keratinocyte differentiation. We demonstrate that OA activates p38 delta but not other p38 isoforms. p38 delta activation is increased as early as 0.5 h after OA addition, and activity is maximal at 8 and 24 h. ERK1 and ERK2 activity are reduced on an identical time course. We show that p38 delta forms a complex with ERK1/2, and overexpression of p38 delta inhibits ERK1/2 activity without reducing ERK1/2 level. Thus, p38 delta may directly suppress ERK1/2 activity. Additional studies show that p38 delta is expressed in the epidermis, suggesting a role for p38 delta in regulating differentiation. To evaluate its function, we show that increased p38 delta activity is associated with increased levels of AP1 and CAATT enhancer binding protein factors, increased binding of these factors to the involucrin (hINV) promoter, and increased expression. Moreover, these responses are maintained in the presence of SB203580, an agent that inhibits p38 alpha and beta, further suggesting a central role for the p38 delta isoform. Dominant-negative p38 also inhibits these responses. These unique observations suggest that p38 delta is the major p38 isoform driving suprabasal hINV gene expression and that p38 delta directly regulates ERK1/2 activity via formation of a p38 delta-ERK1/2 complex.