We have previously demonstrated the requirement of two T helper (Th) populations for the expression of plaque-forming cells (PFC) that bear the dominant cross-reactive idiotype (CRI) associated with the phenyltrimethylammonium (TMA) response (1). In addition to the classic major histocompatibility complex-restricted Th cell, the response was also dependent upon the so-called second order Th2 population, which binds to idiotypic determinants, is carrier specific, but does not require hapten linked to carrier for function. This cell type can be replaced by supernatant (Sn) media from concanavalin A (Con A)-stimulated naive spleen cells. This report involves the study of the Con A Sn derived factor(s) responsible for the expression of CRI bearing PFC populations. When the Brucella abortus (BA)-trinitrophenol (TNP) conjugated antigen is added to TNP-ovalbumin-primed A/J-derived spleen cells in culture, anti-TNP PFC are generated of which only less than or equal to 5% bear the CRI normally associated with anti-TMA antibodies. Upon addition of Con A Sn, the total number of generated anti-TNP PFC doubles, whereas the percentage and number of CRI+ PFC increases approximately eightfold to 10-fold. The factor(s) responsible for this activity are T cell derived, bear Jk serologic determinants, and can be detected in the Sn as early as 4 hr after Con A stimulation. The material appears to be late acting, because it can augment the CRI+ anti-TNP response when added as late as 24 hr before termination of the cultures. In addition, the factor(s) can be bound to and eluted from CRI+ anti-TMA and anti-TNP monoclonal antibodies coupled to Sepharose 4B beads, but not from their CRI- counterparts (i.e., CRI- anti-TMA and anti-TNP antibodies), nor from A/J normal mouse immunoglobulin-coupled beads. Most interestingly, the factor(s) also bind to and can be eluted from the TMA ligand coupled to Sepharose 4B, but not from TNP-Sepharose conjugates. All of these results are consistent with the support the contention that the factor(s) is derived from a Th2-like subpopulation. As assayed by standard protocols, the isolated material contains no T cell replacing factor, interleukin 2, or B cell growth factor activity.