• We examined the correlation between radioactive assay and trephine-disc bioassay of penicillin G sodium, cefamandole nafate, and gentamicin sulfate in ocular tissues of pigmented rabbits after subconjunctival administration of antibiotic. We devised a technique whereby a single sample of tissue could be assayed by both methods. This was achieved by performing the bioassay first, then measuring the residual radioactivity in the agar and specimens. The results of both methods were generally within 13%. An exception was gentamicin in iris and choroidretina, for which the bioassay result was strikingly less than the radioassay value. No such discrepancy was evident when similar studies were carried out with gentamicin in albino rabbits. This suggests that the phenomenon is due to tight binding of gentamicin by melanincontaining tissues. The trephine-disc bioassay provides an accurate measure of diffusible bioactive antibiotic in ocular tissues.