The Heymann nephritis antigenic complex (HNAC) consists of two components, i.e., 1) gp330, a large glycoprotein localized in coated pits of the proximal tubule and glomerular epithelium, and 2) a 44-kDa protein which is homologous to the human alpha 2-macroglobulin receptor-associated protein (RAP). To examine the biosynthesis and assembly of HNAC, tissue fragments prepared from collagenase-digested 1-day-old rat kidneys were radiolabeled, and gp330 and RAP were immunoprecipitated with specific antibodies. By electron microscopy the tubule organization was seen to be largely intact. Results obtained on the biosynthesis of a control brush border protein, dipeptidylpeptidase IV (DPPIV), showed that tubules prepared in this manner are capable of synthesis and posttranslational processing of brush border membrane proteins and thus are suitable for short-term (< 3 h) biosynthetic experiments in vitro. Results of pulse chase and digestion with endoglycosidase H (Endo H) indicated that the time required for newly synthesized gp330 to mature in the endoplasmic reticulum (ER) and transit the middle Golgi compartments [half time (t1/2) = 90 min] was significantly longer than that of DPPIV (t1/2 = 20 min). Coprecipitation and cosedimentation (sucrose velocity gradient centrifugation) experiments showed that gp330 associates with RAP very early after synthesis and that the 44-kDa protein remains associated with gp330 during its subsequent folding, oligomerization, and transport to the Golgi. These findings demonstrate that HNAC assembles in at least two steps. The first step is the association of gp330 with RAP forming a large (19.3S) heterodimer, which sediments with the thyroglobulin (mol wt = 669,000) standard. This step begins within 30 min of synthesis and is Ca2+ dependent. The second step, which occurs > 60 min after synthesis, is the formation of a larger heterooligomer, which results in a shift in size of the complex from 19.3 to 38.6S. Both steps occur before acquisition of Endo H resistance. These results indicate that HNAC consists of a large multimeric complex that is assembled in the rough ER.