Previous studies have used selective neurochemical markers or retrograde tracers to localize the cells in the brain giving rise to efferents to the turtle retina. Because of the relative selectivity of the neurochemical markers or the lack of sensitivity of the previously employed retrograde tracers, these studies did not locate all the efferent cell bodies, or they could not describe the anatomy of the efferent cells. In the present study, cholera toxin B was used as a highly sensitive retrograde tracer to investigate the distribution, number, and morphology of the retinal efferent or centrifugal cell system in turtle brain. Previous studies of the turtle retina have indicated that nitric oxide synthase may be found in some retinal efferents. Therefore, we also did colocalization studies of the retrograde tracer with reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry to investigate nitric oxide as a possible transmitter used by efferent fibers and to localize these NADPHdiaphorase-positive efferent cell bodies in the turtle brain. We found that each eye received projections from approximately 40 efferent cell bodies that were located primarily in the contralateral midbrain. The majority of efferent cell bodies were centered in the isthmic tegmentum; other efferent cells extended more rostrally into the substantia nigra, and some efferent cells extended more caudally into the nucleus raphes superior. The double-label results showed that 30% of the cholera toxin B-like immunoreactive cells were also positive for NADPH-diaphorase. The location of these double-labeled cells around the locus coeruleus corresponded to the NADPH-diaphorase-positive efferent cells in the avian isthmo-optic field. The localization of NADPH-diaphorase in these efferents indicated that they may use nitric oxide to modulate retinal function.