A novel homologue of p38 MAP kinase, called SAPK4, has been cloned which shares 61% amino acid identity with p38 and is expressed predominantly in testes, pancreas and small intestine. We also cloned an alternative form of p38β, termed p38β2, which lacks the additional 8 amino acid insertion unique to p38β. p38, p38β, p38β2, ERK6/p38γ/SAPK3, and SAPK4 were characterized with respect to stimulus-dependent activation in transfected cells, substrate specificity, and sensitivity to inhibition by pyridinyl imidazoles. All homologues were stimulated, although to differing extents, by IL-1β, TNF, sorbitol, and UV. Only SAPK3 and SAPK4 were stimulated significantly by PMA. p38β showed the weakest activation overall. MBP, ATF-2, and both MAPKAP kinase-2 and kinase-3 were good substrates of p38 and p38βin vitro.In contrast, only MBP, ATF2, and MAPKAP kinase-3 proved to be significant substrates of SAPK3 and SAPK4, and of these three, MAPKAP kinase-3 was by far the weakest. p38β had very poor kinase activity for all substrates except MBP. While both p38 and p38β2 were comparably inhibited by SB 203580 and SB 202190, neither SAPK3 nor SAPK4 were inhibited. p38β was partially inhibited by both inhibitors. These data suggest that SAPK3 and SAPK4 form a distinct subset of the p38 MAP kinases with different expression pattern, response to stimuli, substrate specificity, and inhibitor sensitivity.