Isolation of distinct subpopulations of density-fractionated normal human B lymphocytes reveals that the requirements for up-regulation of the vitamin D receptor (VDR) and initiation of 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3]-mediated genomic trans-activation are dependent upon the state of cellular activation. The kinetics of the response differ widely among these B cell subpopulations. However, these density-fractionated B cell subpopulations are phenotypically diverse and therefore are not representative of distinct stages of B cell maturation and differentiation. To examine the role of B cell differentiation on the induction and maintenance of biological receptivity to 1,25-(OH)2D3, we purified naive, germinal center, and memory B cells based on their expression of CD38 and CD44 surface antigens and surface Ig isotype. These phenotypically defined B cell subpopulations were all found to constitutively express VDR, and all exhibited similar activation requirements and kinetics for initiation of 1,25(OH)2D3-mediated genomic trans-activation. Taken together, these results suggest that defined stages of differentiation in normal B cells are not significant predicators of VDR expression or receptivity to 1,25-(OH)2D3. Rather, the degree of cellular activation, regardless of maturation stage, determines whether the effects of this immunoregulatory hormone will influence a mature B lymphocyte.