The enzyme activity which catalyzes the transfer of palmitic acid from palmitoyl-coenzyme A to sublingual gland mucus glycoprotein has been demonstrated in the detergent extracts of the microsomal fraction of rat sublingual and parotid salivary glands. The acyltransferase activity of this fraction was similar in both types of glands. Further subcellular fractionation performed on sublingual glands revealed that the enzyme is associated with the Golgi-rich membrane fraction. Optimum enzymatic activity for fatty acylation of mucus glycoprotein was obtained using 0.5% Triton X-100, 2 mM dithiothreitol, 25 mM NaF, and 10 mM MgCl2 at a pH of 7.4. Higher concentrations of NaF, MgCl2 and dithiothreitol, however, were inhibitory. The apparent Km of the sublingual glands microsomal enzyme for mucus glycoprotein was 0.55 mg/ml and for palmitoyl-CoA, 3.5 X 10(-5) M. A 15% decrease in the acyltransferase activity was obtained with the reduced and alkylated mucus glycoprotein and it showed no activity towards the proteolytically degraded glycoprotein. The 14C-labeled product of the enzyme reaction gave in CsCl density gradient a band at the density of 1.49 in which the 14C label coincided with the glycoprotein. The 14C label in this glycoprotein was susceptible to deacylation with hydroxylamine, and the released labeled material was identified as palmitate.