Experiments were conducted to examine a possible mechanism of activation of rat liver microsomal glycerol-3-phosphate acyltransferase (GPAT) by insulin. Fractions of Mr 1100 were prepared from hind-limb muscles of rats, which had been given intravenous injections of insulin or saline, by a procedure which involved acidification (pH 3.8) and heating (100 degrees C), followed by chromatography on Sephadex G-25 in 50 mM formic acid. These fractions were shown to modify the activity of microsomal GPAT from the livers of fed rats which had not been treated with insulin. The difference in GPAT activity between microsomes supplemented with the Mr 1100 material and those treated with an equal volume of 50 mM formic acid from before the void volume of the column was determined. Relative to the formic acid control, the Mr 1100 material from saline-treated rats decreased GPAT activity, whereas Mr 1100 material from insulin-treated rats increased GPAT activity and the difference of 0.64 nmol/min/mg microsomal protein was significant (P less than 0.01). Fractions of approximately 3000 Da were found to behave in a similar manner and caused a significant (P less than 0.01) increase in GPAT activity of 0.46 nmol/min/mg microsomal protein. These substances, which stimulate GPAT activity, may be related to the putative insulin mediator substance.