In an effort to characterize the second messenger system for LH release, we have previously identified five calmodulin-binding proteins in rat gonadotropes of M(r) > 205 000, 200 000, 135 000, 60 000, and 52 000. In the present study, we have used a calmodulin overlayer assay combined with Western blotting to determine the molecular identity of three calmodulin-binding proteins in rat gonadotropes: the alpha subunit of spectrin (M(r) > 205 000), caldesmon (M(r) 84 000), and the alpha subunit of calcineurin (M(r) 60 000). The M(r) > 205 000 and M(r) 60 000 components or rat pituitary which bind calmodulin are immunoreactive with spectrin and calcineurin antisera, respectively. Rat pituitary also contains an M(r) 84 000 component, which is immunoreactive with polyclonal sera and monoclonal antibody raised to chicken gizzard caldesmon (M(r) 150 000). Like caldesmon from other sources, the M(r) 84 000 component remains soluble after heat treatment and preferentially binds either filamentous actin or calmodulin, depending on the Ca2+ concentration. The three calmodulin-binding proteins were localized specifically in gonadotropes using indirect immunofluorescence microscopy or by Western-blotting cell fractions enriched for gonadotropes. After differential centrifugation of pituitary homogenate, spectrin immunoreactivity was found associated with the nuclear and secretory granule fractions, whereas caldesmon immunoreactivity was seen in the cytosolic fraction and calcineurin in the cytosolic and nuclear fractions. Although the precise role for these proteins remains unknown, the apparent requirement for calmodulin and the small number of calmodulin-binding proteins in the gonadotrope suggest their involvement in mediating GnRH actions.