Activity of protein phosphatase measured in the absence of divalent cations was decreased by 50% during all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation into the granulocytic phenotype. Treatment of HL-60 cells with ATRA led to a dramatic decrease in the amount of protein phosphatase type 2A (PP2A) protein, whereas that of protein phosphatase type 1 (PP1) protein was relatively constant, as detected by immunoblotting with antibodies specific to PP1 and PP2A. The decreased phosphatase activity may be mainly due to a decrease in the expression of the PP2A protein. The mRNA level of PP2A beta was markedly decreased within 5 h after addition of ATRA, but there was only a slight increase in the mRNA level of PP2A alpha. Selective down-regulation of PP2A beta mRNA clearly preceded the cell differentiation induced by ATRA treatment. Thus, PP2A is down-regulated during ATRA-induced differentiation of HL-60 cells into granulocytes.