The cellular mass of sn-1,2-diacylglycerols, which are intracellular second messengers which activate protein kinase C, were quantitatively determined with an enzymatic assay. The method employed to harvest cultured human skin fibroblasts or human epidermal A431 cells prior to extraction of lipid into chloroform/methanol affected diacylglycerol (DAG) levels. Scraping or trypsinization significantly increased DAG levels. A method was devised to allow reliable and reproducible DAG measurements from adherent cells. The addition of methanol prior to scraping was shown to stop cellular metabolism and to permit accurate quantitation. Importantly, this solvent was compatible with cultures grown on plastic. Using this method, growth conditions which could affect DAG levels were investigated. Changes in the osmolality of the culture medium did not affect the DAG levels of A431 cells; exposure of A431 cells to acidic pH or elevated temperature lowered DAG levels. In contrast to fibroblasts, the total DAG levels of A431 cells continued to increase during serum deprivation. The highest DAG levels, normalized to phospholipids, were observed during the exponential growth phase. This ratio dropped when the cultures reached confluency. These experiments also demonstrated that A431 cells possess higher DAG levels than do normal fibroblasts. The function of DAG in cellular regulation is discussed.