Laser micromass analysis was used to investigate the effect of light on the Ca content of rod outer segments in the isolated retina and eyecup of the toad, Bufo marinus. Isolated retinas were incubated in Ringer for which most of the Ca (normally 97% 40Ca) was replaced with 44Ca, so that release of internal Ca (as 40Ca) could be distinguished from the uptake of 44Ca from the external medium. Continuous illumination produced a decline in outer segment 40Ca content. In bright light, the decrease in 40Ca was sigmoidal, beginning with a delay of at least 4 sec, reaching a maximal rate of 1–2 x 10(8) Ca/rod/sec after 8–16 sec, and declining to zero as the light-releasable pool of 40Ca was exhausted within 1–2 min. Decreases of similar magnitude and time course to those observed in isolated retina were also seen in an eyecup preparation. The rate of light-dependent release was reduced 20- to 60- fold by substitution of external Na with choline or Li. The rate of 44Ca uptake from the external medium into rods was little affected during continuous illumination. Uptake, however, was markedly increased in darkness following light exposure. This Ca re-uptake occurred at a rate approximately 2-fold greater in eyecups than in isolated retinas. We interpret our results to show that light causes an increase in the permeability or transport of Ca across the disk membrane and that the Ca is extruded from the rod via Na/Ca (or Na/Ca-K) exchange across the plasma membrane. Cessation of illumination stimulates the resequestration of Ca back into the disks, and this process is somehow enhanced by the presence of the pigment epithelium.