Glycerol release has been generally accepted as an index of lipolysis in the intact heart. The glycerol moiety of glycerol-3-phosphate (glycerol-3-P) is incorporated into triacylglycerols, which are then hydrolysed with release of glycerol. This study investigates the possibility that glycerol may be derived directly from glycerol-3-P instead of passing through the triacylglycerol pool. The cardiac capacity for hydrolysis of glycerol-3-P into glycerol was determined in homogenates of rat hearts. Glycerol-3-P hydrolysis activity in homogenates increased with decreasing pH. The activity was approximately four times higher at pH 5.0 than at pH 7.2 (0.94 +/- 0.11 and 0.25 +/- 0.03 mumol.g wet weight-1.min-1 respectively). The substrate concentration at which half-maximal glycerol-3-P hydrolysis activity was reached did not significantly differ at pH 5.0 and pH 7.2 (4.2 +/- 1.1 mM and 2.9 +/- 1.0 mM respectively). In the intact heart, the pH and substrate conditions found under ischaemia are favourable for direct conversion of glycerol-3-P into glycerol. The glycerol-3-P hydrolysis activity measured in vitro was sufficiently high to account for glycerol production in the ischaemia heart. However, the lack of a stoichiometric relation between cardiac glycerol-3-P and glycerol levels in ischaemia indicates that production of glycerol cannot be explained solely by hydrolysis.