To examine the role of apolipoprotein A-IV (apoA-IV) in the intracellular trafficking and secretion of apoB, COS cells were cotransfected with microsomal triglyceride transfer protein (MTP), apoB-41 (amino terminal 41% of apoB), and either native apoA-IV or apoA-IV modified with the carboxy-terminal endoplasmic reticulum (ER) retention signal, KDEL (apoA-IV-KDEL). As expected, apoA-IV-KDEL was inefficiently secreted relative to native apoA-IV. Coexpression of apoB-41 with apoA-IV-KDEL reduced the secretion of apoB-41 by approximately 80%. The apoA-IV-KDEL effect was specific, as neither KDEL-modified forms of human serum albumin or apoA-I affected apoB-41 secretion. Similar results were observed in McA-RH7777 rat hepatoma cells, which express endogenous MTP. The full inhibitory effect of apoA-IV-KDEL on apoB secretion was observed only for forms of apoB containing a minimum of the amino-terminal 25% of the protein (apoB-25). However, apoA-IV-KDEL inhibited the secretion of both lipid-associated and lipid-poor forms of apoB-25. Dual-label immunofluorescence microscopy of cells transfected with native apoA-IV and apoB-25 revealed that both apolipoproteins were localized to the ER and Golgi, as expected. However, when apoA-IV-KDEL was cotransfected with apoB-25, both proteins localized primarily to the ER. These data suggest that apoA-IV may physically interact with apoB in the secretory pathway, perhaps reflecting a role in modulating the process of triglyceride-rich lipoprotein assembly and secretion.