The involvement of myelin basic protein (MBP) kinases and ribosomal S6 peptide kinases in sheep platelet signal transduction was investigated. Treatment of platelets with 200 nM 12-O-tetradecanoylphorbol-13-acetate (PMA) led to 5-fold stimulations of cytosolic MBP and S6 peptide kinase activities within 1 min. Immunoblotting analysis of phenyl-Superose-fractionated cytosol from PMA-treated platelets with a panel of mitogen-activated protein (MAP) kinase anti-peptide antibodies revealed that one of the activated MBP kinases was p42mapk. This MAP kinase isoform was also stimulated to a lesser extent (approximately 2-fold) when platelets were exposed to 200 microM platelet-activating factor (PAF) for 3 min. The pathways of PAF-activation of p42mapk also involved a protein kinase C-independent route, since the staurosporin analog compound 3 reduced PAF-induced activation by approximately 30% under conditions in which it inhibited PMA-activation of p42mapk by approximately 80%. Another MAP kinase isoform of 44 kDa, most probably p44erk1, was also detected in platelet cytosol, but it was only marginally modulated in response to PMA or PAF. The predominant PMA- and PAF-activated MBP kinase detected after MonoQ fractionation of platelet cytosol did not appear to correspond to a MAP kinase. MonoQ chromatography of platelet cytosol also resolved two PMA- and PAF-activated S6 peptide kinases, which appeared to coelute on phenyl-Sepharose. Western blotting analysis of the MonoQ fractions with antibodies raised against peptide sequences in the S6 kinases p90rsk and p70S6K revealed immunoreactive proteins of approximately 75 kDa and approximately 95 kDa that coincided with the first S6 peptide kinase peak. These proteins probably corresponded to the 502 and 525 amino-acid-length forms of p70S6K. Only the second peak of S6 peptide kinase activity from MonoQ was appreciably stimulated in response to PAF-treatment of platelets, and this was largely abolished by compound 3. It is more likely that the novel MBP and S6 peptide kinases described here, rather than p42mapk and p70S6K, play a significant role in PAF signal transduction in the platelet.