[3H]-all-trans-Retinol injected intraocularly into rats is processed to [3H]-11-cis-retinal, the visually active retinoid that binds to opsin. After 18 h, virtually all (93%) of the radioactive retinals recovered were in the form of 11-cis-retinal. At earlier times, however, both all-trans- and 13-cis-retinals, the latter being a nonphysiological isomer, were formed. Both of these isomers disappeared concomitant with the formation of 11-cis-retinal. The rise and fall of 13-cis-retinal suggest that this isomer can be converted into 11-cis-retinal either directly or indirectly in vivo and, hence, that the biosynthesis of the latter is nonstereospecific. This hypothesis was verified by showing that in double-labeling experiments [14C]-13-cis-retinol was converted into 11-cis-retinal nearly as well (approximately 70%) as [3H]-all-trans-retinol. These studies show that the biosynthesis of 11-cis-retinal can be nonstereospecific and, hence, that the process may be chemically rather than enzymatically mediated in vivo. In contrast, double-labeling studies with [14C]-9-cis-retinol and [3H]-all-trans-retinol showed that very little, if any, of the 9-cis isomer was processed to 11-cis-retinal in vivo although it did form isorhodopsin. This is consistent with what is known about the relative chemical stabilities of 9-cis-retinoids from model studies. The isomerization of 9-cis-retinoids is much slower than that of their all-trans, 13-cis, or 11-cis congeners. These results are discussed in terms of a possible mechanism for the biosynthesis of 11-cis-retinal in vivo and suggest that the isomerization event need not necessarily be enzyme mediated.