The steroid hormone 1␣,25(OH) 2 -vitamin D 3 (1␣,25(OH) 2 D 3 ) generates biological responses in intestinal and other cells via both genomic and rapid, nongenomic signal transduction pathways. We examined the hypothesis that 1␣,25(OH) 2 D 3 action in chick enterocytes may be linked to pathways involving tyrosine phosphorylation. Brief exposure of isolated chick enterocytes to 1␣,25(OH) 2 D 3 demonstrated increased tyrosine phosphorylation of several cellular proteins (antiphosphotyrosine immunoblots of whole cell lysates) with prominent bands at 42-44, 55-60, and 105-120 Kda. The 42-44 Kda bands comigrated with mitogen-activated protein (MAP) kinase (immunoblotting with anti-MAP kinase antibody) The response occurred within 30 s, peaked at 1 min, and was dose-dependent (0.01-10 nM), with maximal stimulation at 1 nM (three-to fivefold). This effect was specific for 1␣,25(OH) 2 D 3 since its metabolic precursors 25(OH)D 3 and vitamin D 3 did not increase MAP kinase tyrosine phosphorylation. The tyrosine kinase inhibitor, genistein, blocked 1␣,25(OH) 2 D 3 -induced tyrosine phosphorylation of MAP kinase, while staurosporine, a PKC inhibitor, attenuated the hormone's effects by 30%. We have evaluated the ability of 1␣,25(OH) 2 D 3 analogs, which have complete flexibility around the 6,7 carbon-carbon bond (6F) or which are locked in either the 6-s-cis (6C) or the 6-s-trans (6T) shape(s), to activate MAP kinase. Thus, two 6F and one 6C analog stimulated while one 6T analog did not stimulate MAP kinase tyrosine phosphorylation. In addition, 1,25(OH) 2 D 3 , a known antagonist of 1␣,25(OH) 2 D 3mediated rapid responses, blocked the hormone effects on MAP kinase. We conclude that 1␣,25(OH) 2 D 3 and analogs which can achieve the 6-s-cis shape (6F and 6C) can increase tyrosine phosphorylation and activation of MAP kinase in chick enterocytes.