Thyroid hormones may have important long-term effects on cellular Ca2+ handling in the heart. We investigated isolated adult rat cardiomyocytes in a primary culture exposed (T3-cells) or not exposed to (control cells) 10(-8) M triiodothyronine (T3) for 48 h. Northern blot analysis revealed reciprocal alterations in the expression of SERCA2 and phospholamban. The ratio of the SERCA2/phospholamban signal was approximately 10 times higher in the T3-cells as compared with the control cells (P < 0.05). Phospholamban protein content was significantly reduced by 33% but SR-Ca(2+)-ATPase protein content was not significantly altered in T3-cells. These results were associated with functional alterations measured by an inverted microscope equipped to monitor fluorescence at two excitation wavelengths as well as cell shortening by a video edge detection unit. The peak calcium transients as measured by fura-2 acetoxymethyl ester (AM) were increased significantly during stimulation at 0.25 and 0.5 Hz in T3-cells compared with control cells (P < 0.05). The monoexponential decline of the fura-2 transient was significantly faster at all frequencies in the T3-cells as compared with control cells (P < 0.05). Interestingly, we observed blunted responses to both isoproterenol stimulation and post rest potentiation in the T3-cells. The intracellular level of sodium as represented by SBFI-AM was significantly lower in the T3-cells compared with the control cells (P < 0.05). The increased SR-Ca(2+)-ATPase/phospholamban ratio and decrease in phospholamban protein content in T3-treated cells was reflected in a parallel increase of contraction and calcium transients and more rapid Ca2+ reuptake, but the post-rest potentiation and response to isoproterenol were reduced.