Enzyme (Dispase ®) dissociation of murine Peyer's patches (PP) 3 yields a cell population rich in T and B lymphocytes and containing 5-9% macrophages (MØ) as defined by morphology and esterase staining. Cell yields by this method are 3-4 times greated than with conventional mechanical methods and viability of cells always exceeds 95%. The virtual absence of IgA plasma cells (<1%) indicates lack of contamination of the cell preparations with intestinal mucosal cells. Enzymatically dissociated PP cells proliferate in response to both T and B cell mitogens. Differences in patterns of mitogen responsiveness of sequentially obtained PP cell fractions are observed suggesting considerable functional heterogeneity of PP cells. Patterns of IgM and IgA synthesis by PP cells in response to the B cell polyclonal activator LPS differ considerably from spleen cells. The development of methodology for preparation of PP cells containing functionally intact T and B cells and enriched for MØ should permit analysis of the cellular basis for functional specialization of PP. © 1982.