1-Butyrylglycerol (monobutyrin) is a novel angiogenic compound that is synthesized and secreted during the differentiation of 3T3-F442A preadipocytes into adipocytes. To study the regulation of monobutyrin biosynthesis we have developed an assay utilizing the lgycerol kinase enzyme from Cellulomonas to quantitate the levels of this compound in cell-conditioned medium. Analysis of several cultured cell types, including tumor cell lines, indicated that monobutyrin production is detectable only from adipocytes, reaching a steady-state concentration of approximately 1.0 microM in conditioned medium. Monobutyrin synthesis was demonstrated in vitro using [14C]butyryl-CoA with total homogenate or particulate fractions from adipocytes. Similar fractions from non-adipocyte cell lines failed to synthesize monobutyrin. This biosynthetic activity was shown to be distinct by substrate competition studies from the microsomal sn-glycerol-3-phosphate acyltransferase, whose activity is known to increase during adipocyte differentiation. The production of monobutyrin was hormonally regulated, as the addition of epinephrine to adipocytes caused a 10-fold increase in the amount of monobutyrin secreted. These results indicate that monobutyrin synthesis is adipocyte specific, occurs through an apparently novel particulate enzyme system, and is regulated in a hormone-dependent manner. The implications of these results for adipose physiology and angiogenesis are discussed.