Defective anergy is a major cause of failed tolerance and is amenable to therapeutic manipulation. To better define the molecular basis of anergy in B cells tolerized by matrix self-antigen, we used complementary approaches of representational difference analysis (RDA) and microarray to identify genes differentially transcribed in anergic as compared to non-tolerant B cells isolated from a well-characterized murine autoantibody transgenic model. Forty RDA clones representing 16 genes were isolated from receptor-stimulated B cells and independently confirmed as differentially expressed in tolerant cells using custom microarray, dot blotting and/or quantitative PCR. Differential expression was conserved in tolerant cells from two different transgenic founder lineages and from two genetically disparate backgrounds. Prominent among recovered gene fragments were genes encoding multifunctional proteins not previously implicated in B cell biology, but with roles in biologic processes fundamental to the tolerance phenotype, including cell growth, proliferation and differentiation. RDA also identified a novel transcript not previously reported in nucleic acid databases. To further explore dependence on receptor stimulation and to identify additional genes, commercial oligonucleotide arrays were probed with labeled B cell transcripts and analyzed for genes differentially expressed in resting as well as stimulated cells and in both B6 and MRL mouse strains. Arrays identified differential expression of a subset of RDA genes as well as 46 additional genes, including subsets engaged in signal transduction, transcriptional regulation, cell growth and apoptosis. Immunoblotting confirmed differential protein expression for galectin-3 and galectin-1, two interactive members of the galectin family, suggesting a novel role for galectins as regulators of immune tolerance.