In the present study, we took advantage of high-resolution multilaser confocal microscopy to examine the distribution of the alpha-subunit of the guanyl nucleotide binding protein subfamily G(q/11) (G(q/11)alpha). Dispersed cultures of pituitary cells were prepared from female weanling rats, fixed, permeabilized, and then stained with monoclonal antiserum (mouse) to the gonadotrope-specific form of secretogranin (SIIp), which was then tagged with Texas Red. Accordingly, the subpopulation of gonadotropes (approximately 15% of total cells) could be identified against a background of other pituitary cell types. G(q/11)alpha was localized with antiserum made in rabbit, then tagged with fluorescein. Hoechst 33258 nuclear stain was also used in some experiments for topological reference. The data indicate localization of the G(q/11)alpha in a cellular region near the plasma membrane and external to the border of the layer occupied by secretory granules. In the absence of activation, there were an average of six clusters of G(q/11)alpha in a section 1 microm thick and through the center of the cell. This corresponds to an average of 60 clusters per cell, assuming a mean gonadotrope diameter of 10 microm. Following continuous treatment with 0.1 microg/ml Buserelin, a metabolically stable GnRH agonist, the average number of clusters increased to 200/cell after 40 min and remained approximately constant for 120 min. This increase was blocked by the protein synthesis inhibitor, cycloheximide. In response to Buserelin, there was an additional increase in the number of clusters inside the cell in the area occupied by the secretory granules and in the perinuclear area. Prolonged (24 h) treatment with Buserelin, sufficient to provoke the onset of desensitization, did not significantly change total numbers of G(q/11)alpha clusters, although more were located in the peripheral compartment, an increase that occurred at the expense of the cytoplasmic compartment. Redistribution of the G(q/11)alpha family may be functionally significant, because this moiety may be rate limiting at the site of regulation of signal transduction.