P revious studies of human adipose tissue lipoprotein lipase (LPL) have focused on enzyme catalytic activity, and have not measured the LPL protein directly. To study the regulation of the LPL protein, an antibody against purified bovine LPL was used. To demonstrate the specificity of the antiserum, adipose homogenates were Western blotted, and adipocytes were radiolabeled and the cell homogenates immunoprecipitated, yielding a single specific band at 53 kD. Breakdown products of LPL were demonstrated at 35 and 20 kD by Western blotting. An ELISA for human adipose LPL was established, in which LPL was sandwiched between affinity-purified antibody and biotinylated affinity-purified antibody. The standard curves for bovine LPL and human adipose LPL were parallel, and LPL activity correlated strongly with LPL immunoreactive mass. Thus, the bovine LPL standard curve was used to estimate LPL immunoreactive mass from human adipose tissue. The regulation of LPL activity and immunoreactive mass were compared in cultured adipocytes in the presence an absence of insulinlike growth factor-I/somatomedin C (IGF-I), insulin, and fetal bovine serum. IGF-I and a high insulin concentration (70 nM) stimulated only the heparin-releasable (HR) component of LPL activity and immunoreactive mass, and neither IGF-I nor insulin affected LPL specific activity. In contrast, 10% fetal bovine serum stimulated HR activity, HR mass, and cellular extractable (EXT) immunoreactive mass, with no effect on EXT activity. This resulted in a decrease in EXT specific activity in response to serum. The effects of the locally produced nucleosides adenosine and inosine were studied in a similar manner. As with serum, adenosine stimulated HR activity, HR mass, and EXT immunoreactive mass, resulting in a decrease in EXT specific activity. Inosine stimulated an increase in HR activity and HR mass, but had no effect on EXT, and thus did not change LPL specific activity. Thus, a sensitive ELISA for adipose tissue LPL has been developed using a specific, well-characterized antibody. Regulation of human LPL immunoreactive mass was demonstrated in vitro by IGF-I, serum, high concentrations of insulin, adenosine, and inosine. This method will permit further investigations into the regulation of the LPL protein.