Gender has recently been implicated as an important modulator of cardiovascular disease. However, it is not known how gender may specifically influence the Ca²⁺-handling deficits that characterize the depressed cardiac contractility of human heart failure. To elucidate the contributory role of gender to sarcoplasmic reticulum (SR) Ca²⁺cycling alterations, the protein levels of SR Ca²⁺-ATPase (SERCA), phospholamban, and calsequestrin, as well as the site-specific phospholamban phosphorylation status, were quantified in a mixed gender population of failing (n=14) and donor (n=15) myocardia. The apparent affinity (EC₅₀) and the maximal velocity (V_max) of SR Ca²⁺-uptake were also determined to lend functional significance to any observed protein alterations. Phospholamban and calsequestrin levels were not altered; however, SERCA protein levels were significantly reduced in failing hearts. Additionally, phospholamban phosphorylation (serine-16 and threonine-17 sites) and myocardial cAMP content were both attenuated. The alterations in SR protein levels were also accompanied by a decreased V_maxand an increased EC₅₀(diminished apparent affinity) of SR Ca²⁺-uptake for Ca²⁺in failing myocardia. Myocardial protein levels and Ca²⁺uptake parameters were then analyzed with respect to gender, which revealed that the decreases in phosphorylated serine-16 were specific to male failing hearts, reflecting increases in the EC₅₀values of SR Ca²⁺-uptake for Ca²⁺, compared to donor males. These findings suggest that although decreased SERCA protein and phospholamban phosphorylation levels contribute to depressed SR Ca²⁺-uptake and left ventricular function in heart failure, the specific subcellular alterations which underlie these effects may not be uniform with respect to gender.