Microsomal triglyceride transfer protein (MTP) is a heterodimer consisting of protein disulfide isomerase and a unique large subunit. Recent studies showing that an absence of MTP is a cause of abetalipoproteinemia indicate that MTP is required for the assembly of very low density lipoproteins in the liver and chylomicrons in the intestine. In this study, complementary DNA encoding the large subunit of hamster MTP was cloned. The cDNA sequence was used to design a 50-base pair oligonucleotide probe for a solution hybridization assay to quantitate MTP large subunit mRNA levels in a study of MTP regulation in male Syrian Golden hamsters. In animals fed a low fat diet, MTP exhibited a proximal to distal gradient of expression in the intestine. MTP activity and large subunit mRNA levels in the liver were about 25 and 10% that found in the proximal intestine, respectively. To investigate the effect of diet on MTP, hamsters were maintained for 31 days on one of four diets: 1) control low fat, 2) high fat, 3) low fat, high sucrose, or 4) diet 1 followed by a 48-h fast. The high fat diet increased MTP large subunit mRNA levels in the liver and throughout the small and large intestine. A 55 and 126% increase was observed in the liver and intestine (duodenum and jejunum), respectively. A 40% increase of intestinal MTP protein mass was also observed. The high sucrose diet caused a significant 55% increase in hepatic MTP mRNA levels but did not significantly affect the intestinal mRNA levels. MTP mRNA levels were unchanged in response to fasting. A short term dietary study showed that intestinal MTP mRNA was up-regulated within 24 h after initiating a high fat diet. An acute hepatic response was not observed. The regulation of MTP mRNA levels by high fat diets was compared to that of the liver fatty acid binding protein (L-FABP) and apolipoprotein B (apoB). ApoB mRNA levels were not significantly affected by a high fat diet. Although L-FABP mRNA levels were increased in the liver and intestine, the onset of the changes did not parallel that of MTP. These results suggest that L-FABP, apoB, and MTP, three proteins which play important roles in the transport of fatty acids and triglyceride in the liver and intestine, are not coordinately regulated by diet in hamsters.