It was found that the Type 2 thymus-independent (TI-2) antigens bacterial levan, trinitrophenyl-Ficoll, and pneumococcal carbohydrate vaccine (PnC) stimulate clonal expansion and antibody secretion in splenic fragments from either hemocyaninprimed or unprimed irradiated recipients bearing B cells from unprimed donors. The in vitro stimulation with TI-2 antigens leads to the expression of isotype switching and provides a more balanced variety of isotypes than is usually observed in vivo. Still, some characteristic patterns of isotypes expressed in vivo to either TI-2 or thymus-dependent (TD) antigens are preserved in vitro. Frequencies of phosphocholine (PC)-reactive B cells responding to either PnC or to PC-hemocyanin (PC-Hy) suggest an appreciable overlap in populations responding to these TI and TD forms of antigen. The existence of a population responsive to either form of PC determinant is supported by the observation that many clones arising in the presence of both forms of antigen express patterns of isotypes that appear as summations of those distinct patterns shown by clones responding to only one form or the other. These data suggest that PC-Hy- and PnC-responding cells may derive from a linear rather than a branched pathway of B cell development and that expression of isotype switching over the lifetime of a developing B cell clone may be regulated in a manner dependent on the form of the stimulating antigen.