Teleost cone inner segments elongate and contract in response to light and circadian signals. Previous studies have shown that teleost cone contraction is triggered by light or dopamine, while cone elongation is triggered by darkness or experimental elevation of cAMP. We have developed procedures for isolating and purifying motile cone fragments consisting of inner and outer segments (CIS-COS) to permit more detailed analysis of light and dopamine regulation of cone retinomotor movements. When retinas are dissected from long-term dark-adapted fish, CIS-COS break off at the base of the ellipsoid and remain attached to the RPE. CIS-COS can be detached from the RPE by brief protease treatment, thereby generating a highly enriched CIS-COS suspension. CIS-COS retain normal morphology and extend new myoids when cultured in darkness or in light plus forskolin, an activator of adenylate cyclase. The microtubule and actin cytoskeletons of the new myoids resemble those of intact cone myoids in vivo. Light inhibits CIS-COS myoid elongation, suggesting that light reception by the outer segment can directly influence cone motility. In dark-cultured CIS-COS, myoid elongation is inhibited half-maximally by nanomolar concentrations of dopamine, suggesting that dopamine effects on motility are mediated by D2-family receptors present on the cone inner and/or outer segment. After dark-induced elongation in culture, CIS-COS myoids can be induced to contract by subsequent culture in the light or with dopamine. Thus isolated cone inner and outer segments possess sufficient cytoskeletal and regulatory machinery to exhibit light- and dopamine-regulation retinomotor movement similar to that observed in intact cones in situ.