Gram-positive cocci frequently contaminate donor corneal tissue and represent the most common cause of postkeratoplasty endophthalmitis. Although gentamicin is currently added to corneal storage medium in an effort to decrease bacterial contamination of donor tissue, it has poor or variable in vitro activity against many strains of streptococci and staphylococci. To investigate whether the antibiotic supplementation of corneal storage media could be improved, we surveyed 11 antibiotics for antimicrobial efficacy under simulated storage conditions against gentamicin-resistant strains of Staphylococcus aureus, S. epidermidis, Streptococcus pneumoniae, and St. viridans. All antibiotics showed markedly reduced activity at 4 C as compared to their predicted activity at 37 C. Bactericidal activity of streptomycin and tobramycin was enhanced by preceding 4 C storage with a three-hour period at room temperature (23 C). Under these conditions, streptomycin showed the best antimicrobial activity of the 11 antibiotics tested. Addition of gentamicin to streptomycin resulted in further improvement of activity against S. aureus and S. epidermidis, whereas the addition of penicillin G to streptomycin enhanced the activity against St. viridans. Optimal antibiotic activity (99% or more killing) against all four isolates of gentamicin-resistant gram-positive cocci was best achieved with the combination of gentamicin, streptomycin, and penicillin G, coupled with a three-hour period at room temperature before 4 C storage.