The present studies were carried out to further characterize the polyclonal B cell activating properties of bacterial peptidoglycan (PG) and to determine if this ubiquitous agent induces in vitro IgM rheumatoid factor (RF) production by lymphocytes from healthy volunteers. Peripheral blood mononuclear cells (PBMC) were cultured in the presence of peptidoglycan, pokeweed mitogen (PWM), a standard polyclonal B cell activator, or additional culture medium. Supernatants were harvested on days 7-8 for determination of total IgM, total IgG, and IgM RF by an enzyme-linked immunosorbent assay (ELISA). PG and PWM induced comparable amounts of total IgM production but PG was a less potent stimulant of total IgG production. PG induced in vitro IgM-RF production in 9/33 experiments, a frequency of response of less than that observed in corresponding PWM stimulated cultures (22/33 experiments). PG-induced IgM-RF production depended upon active protein synthesis and did not correlate with the magnitude of PG-induced total IgM production. The latter finding suggests that PG-induced IgM-RF may not merely reflect polyclonal B cell activation. These results add to a growing list of PG's functional properties and provide further impetus for considering this ubiquitous agent as a potential stimulant for in vivo RF production.