A method for long-term intravitreous microdialysis was used to measure endogenous reduced ascorbic acid in the vitreous of rabbits by HPLC-ECD before and after exposure to intense visible light in the presence of fluorescein. Cellulose microdialysis probes were implanted into the vitreous humor of each eye and after stabilization ascorbic acid measurements were recorded over a 14 day period. Under this experimental condition, normal ascorbic acid concentrations in vitreous varied from 98.0 +/- 9.8 to 106.9 +/- 20.3 microM(mean +/- S.D.). The eyes received light irradiation (25 000 lux) for 2 hr and fluorescein was used as the photosensitizer once or twice. No immediate effects on ascorbic acid concentrations could be observed in the eyes irradiated twice without fluorescein i.v. injections and in the twice fluorescein injected without irradiation. However, in the eyes irradiated once with fluorescein (30 mg kg(-1)), ascorbic acid concentration after irradiation significantly decreased from day 2 and continued over a period of 10 days compared with that before irradiation and maximal reduction was 32.6% (P < 0.005) on day 6 after irradiation. By day 13, the ascorbic acid concentration returned to control levels (P > 0.01). In the eyes irradiated twice with fluorescein injections, ascorbic acid concentration after irradiation decreased even more over the experimental period and the maximal reduction was 65.5% (P < 0.005) on day 5 after irradiation and did not recover over the next 9 days. In the eyes irradiated twice with fluorescein injections plus administration of ascorbic acid (150 mg kg(-1)) 30 min before irradiation, a significant increase (52.5%) of ascorbic acid (P < 0.005) was found on day 1 and control levels of ascorbic acid were maintained from day 2 onward. The protective role of ascorbic acid in the vitreous humor against photodynamic reaction is suggested.