We have investigated the relation between an XO-kDa protein synthesized in vitro in protein-synthesizing system programmed with human liver mRNA [Olofsson, S.-O., Elias, P., Bostrom, K., Lundholm, K., Norfeldt, P.-I., Wiklund, O., Fager, G., and Bondjers, G. (1983) Five monoclonal antibodies directed against LDL-2 as well as polyclonal antibodies against a narrow density cut of LDL-2 (d= 1.030 -1.055) were used to precipitate apoB-related proteins synthesized in vitro in a proteinsynthesizing system programmed with human liver mRNA (or total RNA fraction). With all monoclonal antibodies as well as the polyclonal antibodies, a protein with an estimated molecular mass of 80 f 1.3 kDa (mean f SD, n = 12) could be precipitated. The observation that all monoclonal antibodies used reacted with a p~B~~~~ indicates a close immunological relation between this 80-kDa protein and apoB75kDd. Limited proteolysis of the 80-kDa protein (synthesized in the presence of [35S]-methionine) with Staphylococcus uureus V8 protease generated six [3'S]-methionine-containing bands that could be separated on a polyacrylamide gradient gel (12 -20 %). All these radioactive bands corresponded to major protein-stained bands obtained after limited proteolysis of This observation suggests a structural relation between the two proteins. Taken together, our results indicate that a protein corresponding to apoB'ISkDa is synthesized in vitro in a protein synthesizing system programmed with human liver mRNA (or total RNA fraction).We have also compared a p~B~~~~~ and the major component of apoLDL-2, apoBlOO [Kane, J. P., Hardman, D. A,, and Paulus, H. E. (1 980) Proc. Nut1 Acad. Sci USA 77,2465 -24691 by immunochemical methods. We could demonstrate that six monoclonal antibodies directed against four to six different epitopes on LDL-2, as well as polyclonal antibodies to apoBIOO and a p~B~~~"~, all reacted with a p~B~~~~~ and apoB100. These observations indicate a close immunological relation between the two proteins. Taken together our results support the hypothesis that apoBlOO has a subunit structure. We therefore suggest that apoB75kDa is a subunit of apoBlOO synthesized in human liver.