A c-myc epitope-tagged human dopamine D1 receptor (c-myc D1 receptor) was expressed in Sf9 cells and its cellular distribution under basal conditions and after exposure to the agonist dopamine was examined. In the basal state, immunofluorescently labeled c-myc D1 receptors imaged by confocal microscopy appeared as a bright ring of label predominantly on the cell surface, and to a lesser extent as intracellular clusters of label. This pattern of receptor distribution was confirmed by radioligand-binding assays on plasma membrane and light membrane fractions using the D1 receptor-antagonist [3H]-SCH-23390. After exposure to dopamine, c-myc D1 receptors were redistributed on the cell surface, changing from a continuous ring to a discontinuous pattern of label. Analysis of fluorescence intensity and three-dimensional computer reconstruction of labeled receptors revealed a 30% decrease in surface labeling with no decrease in total number of receptors confirmed by radioligand-binding analysis. These findings constituted the first direct evidence of agonist-induced D1 receptor internalization. The results showed that the combination of confocal microscopy and three-dimensional reconstruction can be used to visualize and assess receptor distribution in Sf9 cells.