This study was performed to reexamine the effect of cholera toxin on total and intracellular alkaline lipoprotein lipase (LPL) activity in rat heart. In addition, the relationship between intracellular triacylglycerol (TG)lipase activity and TG content of cardiac tissue was determined in cholera toxin treated rats. One intravenous injection of cholera toxin increased total LPL activity significantly above control activity 4 h following treatment. After 16 h, total enzyme activity in hearts of cholera toxin treated rats was 2.4-fold above control levels and remained significantly above the control activity up to the 24-h time point. Intracellular alkaline TG lipase activity was increased 24%, 59%, 2.1-fold, and 2.1-fold above control levels measured 0.5, 8, 16, and 24 h following cholera toxin treatment, respectively. Heart TG content fell significantly following cholera toxin treatment, with a maximal reduction seen 8 h following agent injection. At that time, TG was 0.61 μmol/g, a reduction of 63% below the control concentration of 1.8 μmol/g. A negative relationship between myocardial intracellular TG lipase activity and TG concentration of r = −0.83 was highly significant (P < 0.001). These findings indicate that cholera toxin injection can increase total cardiac LPL activity and show that 70% of this increased activity is in the intracellular fraction. The highly significant relationship between enzyme activity and TG content support our working hypothesis that the intracellular alkaline TG lipase (LPL) is playing a role in regulating cardiac TG content.