Very low-density lipoprotein (VLDL) particles are formed in the endoplasmic reticulum (ER) through the association of lipids with apolipoprotein B (apoB). Microsomal triglyceride transfer protein (MTP), which transfers lipid molecules to nascent apoB, is essential for VLDL formation in ER. However, little is known of the distribution and interaction of MTP with apoB within ER. In this study, distribution patterns of apoB and MTP large subunit (lMTP) within ER were examined. Microsomes prepared from HuH-7 cells, a human hepatoma cell line, were further fractionated into rough ER (RER)-enriched subfractions (ER-I fraction) and smooth ER (SER)-enriched subfractions (ER-II fraction) by iodixanol density-gradient ultracentrifugation. ApoB was evenly distributed in the ER-I and the ER-II fractions, while 1.5 times more lMTP molecules were present in the ER-I fraction than in the ER-II fraction. lMTP and apoB were coprecipitated both in the ER-I and in the ER-II fractions by immunoprecipitation whenever anti-apoB or an anti-lMTP antibodies were used. ApoB-containing lipoprotein particles showed a lower density in the ER-II fraction than those in the ER-I fraction. From these results, it is suggested that MTP can function in both rough and smooth regions of ER in human hepatoma cells.