The composition of meibomian gland lipids suggested that fatty acid chain elongation might play a major role in the synthesis of such lipids. A fatty acid synthase preparation from the bovine meibomian gland catalyzed the formation of C16 acid and the enzyme was immunologically quite similar to that in the mammary gland. The microsomal fraction from the gland, on the other hand, catalyzed elongation of endogenous fatty acids in the presence of ATP and Mg2+ and of exogenous C18-CoA using malonyl-CoA and NADPH as the preferred reductant. The elongated products, ranging up to C28 in chain length, were found mainly as CoA esters and products derived from them. With C18-CoA as the exogenous primer, the elongation rate was linear with incubation time up to 20 min but the rate changed in a sigmoidal manner with increasing protein concentration. The elongation rate was maximal at a pH around 7.0. Typical Michaelis-Menten-type substrate saturation patterns were observed with both malonyl-CoA and NADPH. From linear double-reciprocal plots, the Km values for the two substrates were calculated to be 52 and 11 microM, respectively, with a V of about 340 pmol min-1 mg protein-1 with respect to malonyl-CoA. Exogenous CoA esters of C16 to C22 fatty acids were elongated to give products up to C28 without exhibiting any preference for the primer. The present elongation system could account for the formation of most of the very long chains found in meibomian lipids.