In early experiments Ah receptor appeared to be localized in cytosol when in its unoccupied state and it was thought that the receptor translocated into nuclei only when occupied by its ligands. However, a recent report [Whitlock and Galeazzi (1984) J. Biol. Chem. 259, 980-985] concluded that unoccupied Ah receptor in the intact cell was primarily located within the nucleus and that apparent 'cytosolic' Ah receptor was a redistribution artifact caused by fractionation of cells in large volumes of buffer. We examined the effect of buffer volume and ionic strength on apparent 'cytosolic' versus 'nuclear' distribution of unoccupied Ah receptor in liver from C57BL/6J mice and Sprague-Dawley rats as well as Hepa-1c1c9 cells in culture. In all three systems the Ah receptor appears to shift out of the nuclear fraction and into the cytosolic fraction as the volume of buffer is increased or when the ionic strength of the buffer is increased. In each system, however, the distribution of the Ah receptor was identical to the distribution of each of three standard cytosolic marker enzymes: aldolase, glucose-6-phosphate dehydrogenase and lactate dehydrogenase. Co-distribution of unoccupied Ah receptor with these cytosolic marker enzymes during fractionation at varied buffer volumes and ionic strengths makes it seem unlikely that the unoccupied receptor is predominantly a 'nuclear' component in intact cells. Marker enzyme data favor an interpretation that unoccupied Ah receptor is primarily cytoplasmic or that this soluble protein is in equilibrium between cytoplasm and nucleus.