Lipid signaling pathways are thought to play a prominent role in transducing extracellular signals into contractile responses in cardiac muscle. Two putative lipid messengers, diacyglycerol and arachidonic acid, can be generated via distinct phospholipases in separate signaling pathways, but certain stimuli cause them to be elevated in parallel. We tested the hypothesis that these lipids function as comessengers in ventricular myocytes by activating protein kinase C (PKC). In previous work, we demonstrated that the diacylglycerol analog dioctanoylglycerol (diC₈) can be stimulatory or inhibitory toward myocyte twitches depending on how it is applied. Here we report that arachidonic acid and other cis-unsaturated fatty acids (UFA), at concentrations too low for direct effects, synergistically enhance the stimulatory effects of diC₈ and convert inhibitory effects of diC₈ into stimulation of myocyte twitches. Intracellular Ca²⁺ transients changed in parallel with twitch amplitude, suggesting regulation of Ca²⁺ homeostasis by these lipids. cis-UFA also interacted synergistically with the PKC activator phorbol 12-myristate 13-acetate to promote positive inotropic responses. Responses were blocked by the PKC antagonists chelerythrine chloride, bisindolylmaleimide, and Gö-6976. DiC₈ and arachidonic acid also synergistically translocated PKC- and PKC- in intact myocytes. We propose that PKC integrates diacylglycerol and cis-UFA signals in the heart, resulting in preferential activation of positive inotropic mechanisms.