Biosynthesis of the alpha', alpha, and beta subunits of the soybean 7S storage protein (conglycinin) was studied by in vivo and in vitro experiments. One hour in vivo labeling produced polypeptides of 72 kilodaltons (kD) and 78 kD which, in a subsequent 1-h chase period, gave rise to a heterodisperse band of polypeptides of 76-83 kD, the apparent molecular weights of mature alpha and alpha', respectively. After a 3-h chase period only mature alpha and alpha' were labeled. The pre-alpha' (80 kD) and pre-alpha (78 kD) polypeptides produced by in vitro translation of total seed poly(A)+ RNA did not coelectrophorese with polypeptides labeled in vivo. The mature beta-subunit (53 kD), produced in vivo during the 1-h chase period, apparently was translated as a 50 kD polypeptide in vitro. The data suggest that, in vivo, primary translation products undergo both cotranslational and posttranslational modifications during the formation of mature subunits. cDNA clones complementary to soybean seed poly(A)+ RNA were prepared and subsequently identified by hybrid-release and immunoprecipitation experiments. Clone pGmc 236 (550 base pairs) (bp) was shown to contain sequences complementary to mRNAs for the alpha', alpha, and beta subunits on the basis of hybrid-release experiments, corroborating tryptic fingerprints that demonstrate that these subunits are closely related to each other. Although selected by a single cDNA clone, the mRNAs are not identical because the alpha' mRNA was eluted from the filter-bound cDNAs at a lower temperature than were the alpha beta mRNAs, pGmc 236 hybridized on Northern blots to mRNAs of approximately 2500 nucleotides, sufficient size to code for the alpha' or alpha subunit of the 7S storage protein.