We compared the growth patterns of gram-negative bacilli in the vitreous humor (experimental endophthalmitis in rabbits) and in a subcutaneous site of infection (croton oil pouches in rats). In untreated animals, inoculation of Klebsiella pneumoniae or Pseudomonas aeruginosa into either site was followed by a period of rapid bacterial multiplication. Thereafter, the numbers of bacteria in the vitreous humor fell spontaneously, whereas those in the subcutaneous site remained stable. During treatment with antibiotics, there was a decline in the numbers of bacteria in both sites. However, once the drug had been eliminated, the numbers of bacteria remained low in the vitreous humor but increased in the subcutaneous site. These findings suggest that during the course of infection, there was depletion of an essential bacterial nutrient or accumulation of an antibacterial substance in the vitreous humor but not in the subcutaneous site. To examine some of these possibilities, we made biochemical measurements during the course of untreated infection. In general, the biochemical changes in the two sites were similar except that the pH fell to about 6.6-6.8 in the vitreous humor but remained above 7.0 in the subcutaneous site. None of the biochemical changes that we observed seemed likely to account for the spontaneous decline in bacterial numbers in the vitreous humor. Further study is warranted to determine the cause of the antibacterial effect in the vitreous humor during the course of experimental bacterial endophthalmitis.