Allelic differences are known to influence many important aspects of class II biosynthesis, including subunit assembly, Ii chain associations, and DM-mediated peptide loading. Mutant mouse strains lacking Ii chain expression have been previously studied on mixed genetic backgrounds. The present experiments describe cellular and functional characteristics of congenic BALB/c Ii chain mutants. As expected, class II surface expression was markedly decreased, but in contrast to IA d-transfected cell lines, serological analysis of BALB/c Ii chain-deficient spleen cells gave no evidence for discordant expression of class II conformational epitopes. Thus, we conclude that properly folded class II molecules are exported via the Ii chain-independent pathway. Functional assays demonstrate consistently superior peptide-loading capabilities, suggesting that these IA d molecules are empty or occupied by an easily displaced peptide (s). Defective B cell development was observed for three mutant strains established on diverse genetic backgrounds. Ii chain function is also essential for optimal class II surface expression by mature splenic dendritic cells. Surprisingly, we observe in BALB/c Ii chain mutants, relatively efficient maturation of CD4+ T cells in the periphery and secondary proliferative responses elicited upon peptide challenge. The milder phenotype displayed by BALB/c Ii chain mutants in comparison with class II functional defects previously described for mouse strains lacking Ii chain is likely to have an effect on disease susceptibility.