VH fragments were prepared from several mouse IgM molecules by cyanylation. In all cases VH fragments were purified to homogeneity by using Ig light chain affinity columns. Several different anti-VH antisera were prepared in rabbits and the specificities of these antibodies were studied. Two patterns of cross-reactivities were observed: (a) some anti-VH antibodies reacted only with closely related VH molecules, e.g., anti-VH HPC52 anti bodies reacted only with VH of phosphorylcholine-binding myeloma or hybridoma proteins, and concordantly, stained about 4% of mouse spleen B cells; (b) on the other hand, antisera-like anti-VH 104E and 8916 antibodies were very cross-reactive. Binding assays showed that both of these anti-VH antibodies reacted with 50-60% of mouse immunoglobulins. However, they recognized mainly nonoverlapping populations of mouse immunoglobulins, and thus the pool of these antibodies reacted with about 95% of mouse VH regions. Concordantly, anti-VH 104E antibodies stained in the fluorescence-activated cell sorter (FACS) analysis more than 50% of mouse spleen B cells. Cross-reactive anti-VH antibodies ("anti-framework") did not stain T cells nor did they immunoprecipitate VH-like molecules which were synthesized by T cells.