The existence of the murine fetal liver hybridomas whose Ig phenotype is identical to that of pre-B cells (cytoplasm μ +; cell surface Ig -) has made it possible to study certain structural characteristics associated with the pre-B cell Ig. Previous studies suggested that significant size heterogeneity existed between the μ-chains synthesized by different fetal liver hybridomas. The present study demonstrated that these m.w. differences were apparently not related to different degrees of glycosylation, because treatment with tunicamycin did not alter the observed size variation as determined by SDS gel electrophoresis. Analysis of the cyanogen bromide-generated peptides suggested that the variation did not occur in the Fc fragment and was apparently localized to the V(H) and/or CH 1 domains of the μ-chain. To determine whether the μ-chains synthesized by the pre-B-like hybridomas (i.e., fetal liver hybridomas with the pre-B cell phenotype) were of the secreted or membrane type, the C-terminal structure of the μ-chains was analyzed. Carboxypeptidase-A digestion of purified μ-chains from these hybridomas yielded approximately 1 residue of tyrosine release per μ-chain. These results suggested that the μ-chains synthesized by the pre-B-like hybridomas were of the secreted type, as membrane-type μ-chains failed to release tyrosine under similar conditions of digestion. Furthermore, the C-terminal octapeptide generated by cyanogen bromide cleavage was readily identified in the μ-chains of those pre-B cell hybridomas studied further, indicating that these μ-chains were predominantly of the secreted type.