Cardiac hypertrophy may result from extracellular stimuli activating intracellular signalling pathways, including the mitogen-activated protein kinase (MAPK) pathways. The MAPK pathways include three cascades: ERK1/2, JNK, and p38. ERK1/2 is activated by mitogens whereas stress-related stimuli activate JNK and p38. In cardiac myocytes, activation of ERK1/2 by H₂O₂ is attenuated by p38. This study was to determine if p38 inhibits activation of ERK1/2 by PMA, a mitogen and potent activator of ERK1/2, in adult cardiac myocytes. Inhibition of p38α/β with SB203580 increased PMA-stimulated ERK1/2 and MEK1/2 phosphorylation. FPLC of myocyte lysates on MonoQ revealed two peaks of PMA-stimulated MBP kinase activity, ERK2 and ERK1. PD 98059, which blocks MEK1/2 activation, attenuated PMA-induced ERK1/2 activation. SB203580 did not increase ERK1/2 activity. Three peaks of PMA-stimulated MEK activity were detected following chromatography on Mono Q. Peaks 1, 2, and 3 eluted at 20-25 mM, 80-100 mM, and 210 mM NaCl, respectively. PD 98059 inhibited each of these activities. Peaks 2 and 3 were increased three-fold by SB203580. In addition, a new peak was detected eluting at 40-70 mM NaCl. Hence, in adult cardiac myocytes, p38α/β attenuates PMA-dependent MEK1/2 and ERK1/2 phosphorylation.