The activity of lipoprotein lipase (LPL) in adult rat heart cardiomyocytes after overnight culture on laminin-coated plates for 18-22 h was compared with enzyme activity in freshly isolated cardiomyocytes. LPL activity in cellular homogenates from cultured cardiomyocytes and freshly isolated cells was 240 and 233 nmol oleate released h-1.mg-1 protein, respectively. LPL specific activity (mU/ng LPL protein) was 0.07 in cultured cells compared with 0.42 in freshly isolated cells, indicating an increased content of inactive LPL mass after overnight culture. The heparin-induced release of LPL activity into the medium of cultured cardiomyocytes (198 nmol.h-1.mg-1) was much greater than heparin-releasable LPL (HR-LPL) activity (59 nmol.h-1.mg-1) from freshly isolated cells. HR-LPL activity from cultured cardiomyocytes was dependent on serum (16.3-fold activation) and was inhibited by high ionic strength (1 M NaCl) and by a polyclonal antibody to LPL. Cultured cardiomyocytes also had more immunodetectable LPL on the cell surface compared with freshly isolated cardiomyocytes, consistent with increased HR-LPL activity. Therefore, overnight culture may permit cardiomyocytes time to recover from the stress of isolation by increasing the content of LPL on the cell surface.