D-fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) derived from rabbit muscle facilitated immunoglobulin production by human-human hybridoma HB4C5 cells under serum-free condition. IgM production by HB4C5 cells was stimulated by the addition of aldolase from rabbit muscle at the concentrations of more than 100 mu g ml(-1). The enzyme also enhanced IgM and IgG production by human peripheral blood lymphocytes about 5.3-fold and 1.4-fold, respectively. This fact suggests that aldolase from rabbit muscle stimulates immunoglobulin production not only by the specified hybridoma cell line, but also by unspecified immunoglobulin producers; however, yeast aldolase was ineffective in accelerating immunoglobulin production by HB4C5 cells. The IPSF activity was lost by trypsin digestion at 100 mu g ml(-1); however, limited fragmentation by the lower concentrations of trypsin facilitated the IPSF activity of rabbit muscle aldolase about twofold even though the enzymatic activity was inactivated. These results imply that the immunoglobulin production-stimulating effect of aldolase is irrelevant to its enzymatic function, and the activity was defined as a novel role of rabbit muscle aldolase.