Carbovir is a novel carbocyclic guanosine derivative that has potent in vitro activity against human immunodeficiency virus, the causative agent of acquired immunodeficiency syndrome (AIDS). Two methods of sample preparation were developed for the analysis of carbovir in rat blood. Solid-phase extraction on C18 extraction columns proved to be the most effective. Whole rat blood (200 microliters) was diluted with 0.8 ml of distilled water containing the internal standard. After two freeze-thaw cycles to lyse the red blood cells and subsequent centrifugation at 13,000 g, the supernatant was loaded on the C18 extraction columns. Carbovir and the internal standard were eluted with methanol-water (60:40). The extract was evaporated and reconstituted in mobile phase and the samples were injected onto a high-capacity reversed-phase column. The compounds were detected at 252 nm. Other nucleosides that could be used in the treatment of AIDS such as zidovudine and acyclovir did not interfere. Standard curves were linear over the concentration range 0.156-28.0 micrograms/ml (r2 greater than 0.99). The within-day coefficient of variation was less than 7.6% at all concentrations (n = 4). The between-day coefficient of variation ranged from 16.7 to 2.0% (n = 14). The limit of sensitivity was 0.05 micrograms/ml with a 200-microliters blood sample and the average extraction recovery was 74%. Carbovir was stable in rat blood for at least 4 h at 37 degrees C. The assay was used to determine the blood levels of carbovir in a rat after a 20 mg/kg intravenous dose.