The effects of short (1 min) and long (7-10 min) exposure to hyposmotic solution on excitation-contraction coupling in rat ventricular myocytes were studied. After short exposure, the action potential duration at 90% repolarization (APD₉₀), the intracellular Ca²⁺ concentration ([Ca²⁺]_i) transient amplitude, and contraction increased, whereas the L-type Ca²⁺ current (I_Ca,L) amplitude decreased. Fractional sarcoplasmic reticulum (SR) Ca²⁺ release increased but SR Ca²⁺ load did not. After a long exposure, I_Ca,L, APD₉₀, [Ca²⁺]_i transient amplitude, and contraction decreased. The abbreviation of APD₉₀ was partially reversed by 50 µM DIDS, which is consistent with the participation of Cl current activated by swelling. After 10-min exposure to hyposmotic solution in cells labeled with di-8-aminonaphthylethenylpyridinium, t-tubule patterning remained intact, suggesting the loss of de-t-tubulation was not responsible for the fall in I_Ca,L. After long exposure, Ca²⁺ load of the SR was not increased, and swelling had no effect on the site-specific phosphorylation of phospholamban, but fractional SR Ca²⁺ release was depressed. The initial positive inotropic response to hyposmotic challenge may be accounted for by enhanced coupling between Ca²⁺ entry and release. The negative inotropic effect of prolonged exposure can be accounted for by shortening of the action potential duration and a fall in the I_Ca,L amplitude.