Previous studies from our laboratory have suggested that the assembly of lipoproteins by the liver is not completed in the rough endoplasmic reticulum but continues while the particles are en route to or within the Golgi apparatus. To investigate further the role of the Golgi apparatus in lipoprotein assembly, mice were injected with [3H]glycerol and killed 7.5 to 45 min after injection. Microsomes and Golgi apparatus-rich fractions were isolated from the livers and separated into membrane and content fractions. TG within microsomal and Golgi membranes were labeled, rapidly reaching peak specific activity within 7.5 min of isotope injection. The specific activity of TG in microsomal membranes decreased to approximately 40% of peak values by 45 min, whereas the specific activity of TG in the Golgi membranes decreased to approximately 30% of peak values by 45 min. To determine whether the turnover of the Golgi membrane TG pool was dependent on microsomal TG transfer protein (MTP), mice were gavaged with an MTP inhibitor, and the labeling experiments were repeated. Inhibition of MTP attenuated the turnover of newly synthesized Golgi membrane TG by approximately 50% and the turnover of microsomal membrane TG by approximately 40%. Based on the rapid turnover of the Golgi membrane TG pool and the attenuation of the turnover of this pool by MTP inhibitor, we propose that lipid is transferred from the Golgi membrane to luminal lipoproteins in an MTP-dependent manner. The results support our hypothesis that assembly of VLDL continues within the Golgi apparatus.