Background:Little is known about the ocular penetration following intrastromal or intracameral injection of amphotericin B (AMB), the current drug of choice in fungal keratitis. Concentrations of AMB were investigated in the cornea and aqueous humor of rabbits after using one of three different routes of administration: topical 0.25% AMB eye drops and intrastromal and intracameral injection of AMB (10 µg). Methods: Forty-five healthy rabbits were randomly divided into 3 groups. The eyes of group A and group B received a 0.1-ml intrastromal and intracameral injection, respectively, containing 10 µg AMB. Group C received topical 0.25% AMB (corneal epithelium debrided, every 5 min for 30 min). Cornea and aqueous humor concentrations of AMB after 30 min, 6 h, 1, 3 and 7 days were analyzed by high-performance liquid chromatography. Results: After a single injection, effective drug levels were achieved in corneas in group A, maintained for 7 days, exceeding the minimum inhibitory concentration at which 90% of isolates are inhibited (MIC<sub>90</sub>) for a wide spectrum of fungi and molds. There were significant differences (p < 0.001) compared with group B and group C. Effective drug levels were achieved in the aqueous humor in group B at 30 min after a single injection, exceeding MIC<sub>90</sub>, but drug levels decreased abruptly within 1 day. There were significant differences (p < 0.004) compared with group A and group C, and a considerable amount of AMB was detected in corneas and aqueous in group C within 1 day. Conclusion: High drug levels can be reached that cover the MICs of most fungi in the rabbit cornea and aqueous humor after intrastromal and intracameral injection, respectively. Penetration of topical AMB greatly increased after epithelial abrasion.