The association of alkaline phosphatase (ALPase) with the cytoskeleton in lymphoid cells was investigated. Extracting cells with non-ionic detergents such as Triton, we determined that ALPase is present in the cytoskeletal fraction in fully differentiated B lymphocytes, X63 myeloma cells and Sp2/O hybridoma cells. During the course of B-lymphocyte activation, the ALPase shifted from a soluble to a Triton-insoluble form. Changes in the phosphorylation of Triton-insoluble proteins with molecular weights of 120, 100, 90, 75, 34 and 31 kDa were detected, coinciding with the appearance of the ALPase in this fraction. The possible role of ALPase in the differentiation of B cells is discussed.