Target inactivation analysis was used to measure the functional size of uridine diphosphogalactose: N-acetylglucosamine beta(1,4)galactosyltransferase (galactosyltransferase), cytidine monophospho-N-acetyl-neuraminic acid: beta-galactoside alpha(2,6) sialytransferase (sialyltransferase), and uridine diphosphatase (UDPase) in Golgi membranes isolated from rat liver. The size of nucleoside diphosphatase (NDPase), an enzyme similar to UDPase but localized in rat liver endoplasmic reticulum, was also estimated by target inactivation analysis. The related enzymes, UDPase and NDPase, have target sizes of 96 +/- 4 and 77 +/- 3 kDa, while galactosyltransferase and sialyltransferase have target sizes of 97 +/- 10 and 130 +/- 20 kDa, respectively. The target inactivation sizes of galactosyltransferase and of sialyltransferase are about twice the monomer molecular weights of these enzymes obtained from sedimentation studies of the solubilized membranes as well as those predicted from previously reported cDNA sequences. We conclude from our studies that galactosyltransferase and sialyltransferase probably function as dimers in the Golgi membrane.