The opportunity and ability to photobiosynthesize vitamin D3 by exposing skin to ultraviolet-B (UVB) irradiation from the sun was compared using the nocturnal/crepuscular Mediterranean House Gecko Hemidactylus turcicus and the diurnal Texas Spiny Lizard Sceloporus olivaceous. Texas spiny lizards had a greater opportunity for photobiosynthetic production of vitamin D3 than geckos. This was revealed by vitamin D3 photoproduct production in models (ampoules containing an alcohol solution of vitamin D3 precursor) placed at locations inhabited by free-living lizards at similar times of occupancy. Alternatively, geckos seemed able to maximize their limited photobiosynthetic opportunity with a higher rate of conversion of provitamin D3 to photoproducts. This was revealed by photoproduct conversion in patches of lizard skin exposed to ultraviolet lamps in the laboratory. Stomach-content analysis showed the spiny lizards to have dietary sources of vitamin D3, the geckos may or may not. This is the first documentation that mostly nocturnal geckos may rely on photobiosynthesis of vitamin D3 and that they might have a more sensitive mechanism than diurnal lizards to compensate for their limited exposure to natural UVB radiation. Future studies should investigate sexual, seasonal, age, and species differences in photobiosynthetic opportunity and ability.