Frogs were injected intravitreally with [3H]acetate, and the formation of [3H]-labeled squalene and cholesterol in the retina and their incorporation into rod outer segment (ROS) membranes were evaluated biochemically over a 60-day time course. ROS [3H]squalene specific activity was maximal by 1-3 days, then declined with a half-time of approximately 20-30 days. In contrast, the specific activity of ROS [3H]cholesterol initially increased to a level substantially less than that of [3H]squalene, and then remained constant. Thus, ROS squalene appears to turn over without obligatory conversion to, or coturnover with, ROS cholesterol. When [3H]acetate was injected into one eye, radiolabel in non-saponifiable lipids of the contralateral retina represented < 1% of those recovered from the ipsilateral retina; hence, systemic contributions to de novo synthesis were obviated. Long-term (> or = 8 hr) in vitro incubations of isolated retinas with [3H]acetate resulted in incorporation of [3H]-labeled sterols and squalene into ROS, at levels comparable to those observed in ROS from companion incubated eyecup preparations and from retinas 8 hr after intravitreal injection of [3H]acetate. These results demonstrate that the in vitro system faithfully reflects the in vivo biosynthetic capacity with respect to isoprenoid lipid metabolism, and suggest that de novo synthesis within the neural retina is responsible for generating most, if not all, of the [3H]squalene and [3H]cholesterol formed under the given conditions. Treatment of retinas in vitro with brefeldin A or energy poisons blocked transport of newly synthesized opsin, but not squalene, to the ROS. Furthermore, frogs maintained at 8 degrees C exhibited marked suppression of incorporation of newly synthesized protein into the ROS, while [3H]squalene incorporation was only minimally reduced, compared with frogs maintained at 22 degrees C. These results are consistent with prior findings that suggest that lipids are transported to the ROS by a mechanism distinct and independent from that employed for intracellular trafficking of opsin and other ROS-destined membrane proteins.