1 The present study examined the role of myosin light chain kinase (MLCK), PKC isozymes, and inositol 1,4,5-trisphosphate (IP 3 ) receptor in the positive inotropic effect of a 1 -adrenergic stimulation in atrial myocardium. 2 We measured inotropic effects of phenylephrine (0.3-300 mM) in isolated left atrial preparations (1 Hz, 371C, 1.8 mM Ca 2 þ , 0.3 mM nadolol) from male 8-week FVB mice (n ¼ 200). Phenylephrine concentration-dependently increased force of contraction from 1.570.1 to 2.870.1 mN (mean7s.e.m., n ¼ 42), which was associated with increased MLC-2a phosphorylation at serine 21 and 22 by 67% and translocation of PKCe but not PKCa to membrane ( þ 30%) and myofilament ( þ 50%) fractions. 3 MLCK inhibition using ML-7 or wortmannin right-shifted the concentration-response curve of phenylephrine, reducing its inotropic effect at 10 mM by 73% and 81%, respectively. 4 The compound KIE1-1 (500 nM), an intracellularly acting PKCe translocation inhibitor peptide, prevented PKCe translocation and augmented the maximal inotropic effect of phenylephrine by 40%. In contrast, inhibition of Ca 2 þ -dependent PKC translocation (KIC1-1, 500 nM) had no effect. Chelerythrine, a PKC inhibitor, decreased basal force without changing the inotropic effect of phenylephrine. 5 The IP 3 receptor blocker 2-APB (2 and 20 mM) concentration-dependently decreased basal force, but did not affect the concentration-response curve of phenylephrine. 6 These results indicate that activation of MLCK is required for the positive inotropic effect of a 1 -adrenergic stimulation, that the Ca 2 þ -independent PKCe negatively modulates this effect, and that PKCa and IP 3 receptor activation is not involved.