Recent evidence suggests that κ-opioid receptor (OR) agonists and K(ATP) channel activation exert antihypertrophic effects on cardiac myocytes. We studied the role of K(ATP) channels in the antihypertrophic effects of ORs in primary cultures of neonatal rat ventricular myocytes exposed for 48 h to the α(1) adrenoceptor agonist phenylephrine and the relative contributions of mitochondrial K(ATP) (mitoK(ATP)) and sarcolemmal K(ATP) (sarcK(ATP)). Furthermore, we elucidated the pathway between ORs and K(ATP) channels and their impact on intracellular Ca(2+) ([Ca(2+)](i)) transients. Hypertrophy of cardiomyocytes was characterized by increases in i) total protein content; ii) cell size and iii) [(3)H]leucine incorporation. Phenylephrine (10 μM) increased the three parameters. Trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamid methanesulfonate salt (U50,488H), a selective κ-opioid receptor agonist, prevented phenylephrine-induced hypertrophy and [Ca(2+)](i) transients. The effect of U50,488H was abolished by nor-binaltorphimine, a selective κ-OR antagonist, indicating that the effect was κ-OR-mediated. The protein kinase C inhibitor chelerythrine and the K(ATP) channel inhibitors glibenclamide (50 μM), a nonselective K(ATP) antagonist, and 5-hydroxydecanoic acid (100 μM), a mitochondrial selective K(ATP) antagonist, reversed the antihypertrophic effect of U50,488H, and there was no significant difference between the two K(ATP) channel blockers. Moreover, we also determined the expression of the Kir6.2 subunits of the K(ATP) channel, which increased in response to U50,488H in the presence of phenylephrine, but was suppressed by chelerythrine, glibenclamide and 5-hydroxydecanoic acid. U50,488H also attenuated the elevation of [Ca(2+)](i). This study suggests that K(ATP), and particularly the mitochondrial K(ATP,) mediates the antihypertrophic effects of κ-opioid receptor stimulation via the PKC signaling pathway.