Retinomotor movements include elongation and contraction of rod and cone photoreceptors, and mass migration of melanin-containing pigment granules (melanosomes) of the retinal pigment epithelium (RPE) within the eyes of fish, frogs, and other lower vertebrates. Eyes of these animals do not contain dilatable pupils; therefore the repositioning of the rods and cones and a moveable curtain of pigment granules serve to modulate light intensity within the eye. RPE from sunfish (Lepomis spp.) can be isolated from the eye and dissociated into single cells, allowing in vitro studies of the cytoskeletal and regulatory mechanisms of organelle movement. Pigment granule aggregation from distal tips of apical projections into the cell body can be triggered by the application of underivatized cAMP, and dispersion is effected by cAMP washout in the presence of dopamine. While the phenomenon of cAMP-dependent pigment granule aggregation in isolated RPE was described many years ago, whether cAMP acts through the canonical cAMP-PKA pathway to stimulate motility has never been demonstrated. Here, we show that pharmacological inhibition of PKA blocks pigment granule aggregation, and microinjection of protein kinase A catalytic subunit triggers pigment granule aggregation. Treatment with a cAMP agonist that activates the Rap GEF, Epac (Effector protein activated by cAMP), had no effect on pigment granule position. Taken together, these results confirm that cAMP activates RPE pigment granule motility by the canonical cAMP-PKA pathway. Isolated RPE cells labeled with antibodies against PKA RIIα and against PKA-phosphorylated serine/threonine amino acids show diffuse, punctate labeling throughout the RPE cell body and apical projections. Immunoblotting of RPE lysates using the anti-PKA substrate antibody demonstrated seven prominent bands; two bands in particular at 27 and 64 kD showed increased levels of phosphorylation in the presence of cAMP, indicating their phosphorylation could contribute to the pigment granule aggregation mechanism.