1. The sphingolipid ceramide, a primary building block for all other sphingolipids, is associated with growth arrest, apoptosis, and lipotoxic dysfunction. Interestingly, ceramide may attenuate high glucose-induced myocyte dysfunction, produce Ca2+ influx, and augment smooth muscle contraction. To determine the role of ceramide on cardiac excitation-contraction (E-C) coupling, electrically paced adult rat ventricular myocytes were acutely exposed to a cell-permeable ceramide analog (10 pm-100 microM) and the following indices were determined: peak shortening (PS), time-to-PS, time-to-90% relengthening, and the maximal velocity of shortening and relengthening (+/-dLdt). Intracellular Ca2+ properties were assessed using fura-2AM fluorescent microscopy. 2. Our results revealed a concentration- and time-dependent increase of PS in ventricular myocytes in response to ceramide associated with an increase in +/-dLdt. The maximal increase in PS was approximately 35% from control value and was maintained throughout the first 20 min of ceramide exposure. However, the ceramide-induced increase in PS was not maintained once the exposure time was beyond 20 min. Acute exposure of ceramide significantly enhanced intracellular Ca2+ release, although at a much lower concentration range. The ceramide-induced augmentation of PS was not significantly affected by inhibition of phosphatidylinositol (PI)-3-kinase, protein kinase C (PKC), ceramide-activated protein phosphatase (CAPP), and nitric oxide (NO) synthase. 3. Our data suggest that ceramide acutely augments the contractile function of cardiac myocytes through an alternative mechanism(s) rather than PI-3-kinase, PKC, CAPP, or NO.