Abstract Previously, we reported that human B lymphocytes can be stimulated by trinitrophenylated Brucella abortus (TNP-Ba) in vitro to generate T-independent (TI) hapten specific plaque-forming cells (PFC). Furthermore we showed that addition of Con A on Day 3 of culture enhanced the anti-TNP response. In this report the characteristics of the anti-TNP PFC responses elicited by TNP-Ba in the presence or absence of Con A were further defined by estimating precursor frequencies and clone sizes, and by assessing the diversity of the anti-TNP-PFC response using hapten inhibition profiles. Addition of Con A to TNP-Ba-stimulated cultures was associated with a 2.3- to 3.6-fold increase in anti-TNP precursors and was also accompanied by a more vigorous clonal expansion. Fine specificity analysis of anti-TNP PFC revealed that Con A addition resulted in PFC with unique hapten inhibition profiles in that they were less inhibitable by TNP-EACA, TNP-lysine, and DNP-lysine but more inhibitable by DNP-glycine when compared to PFC generated by TNP-Ba alone. These findings suggest that at least some of the additional precursors recruited in the presence of Con A are qualitatively distinct from those activated by TNP-Ba alone since they express different V region gene products.