Measurements were made of the activities of enzymes of glycerolipid synthesis in homogenates of interscapular brown adipose tissue. The enzymes studied were: mitochondrial and microsomal forms of glycerolphosphate acyltransferase (GPAT), Mg2+-dependant phosphatidate phosphohydrolase (PPH), fatty acyl-CoA synthetase (FAS) and monoacyl-glycerolphosphate acyltransferase (MGPAT). In normal animals cold-exposure (4°C) for 3-days increased all activities relative to tissue DNA. Mitochondrial GPAT showed a particularly marked increase (5-fold). Administration of the adipose tissue specific ß3-adrenergic agonist (BRL-26830-A) resulted in a mimic of cold-induction in microsomal GPAT activity; no change was recorded for the other enzyme activities. Phenylephrine administration, either with BRL-26830-A or alone, resulted in reduction of activities of PPH, mitochondrial GPAT and MGPAT. Diabetes decreased mitochondrial GPAT activity but did not affect the increase in activity due to cold-exposure. Diabetes prevented the increase in activities of PPH and FAS due to cold-exposure. Hypothyroidism mimicked and enhanced the cold-induced increase in mitochondrial GPAT activity but did not affect the activity of PPH. Hyperthyroidism induced an approximate doubling of PPH activity but did not affect mitochondrial GPAT activity. Adrenalectomy resulted in an increase in mitochondrial GPAT activity which mimicked the effect of cold-exposure. PPH activity was increased by adrenalectomy and decreased by cold-exposure in the adrenalectomised animals. Cycloheximide administration prior to cold-exposure reduced the cold-induced increase in mitochondrial GPAT. These findings are relevant to signals that drive early events in mitochondriogenesis and cell proliferation in brown adipose tissue on exposure to cold. Mitochondrial GPAT activity was observed in both outer and inner mitochondrial membranes from liver. Solubilisation of the mitochondrial GPAT activity resulted in an increase in Km for glycerol-3-phosphate and sensitivity to N-ethylmaleimide. The solubilised activity was precipitated and stabilised in 12–15% polyethylene-glycol.