We sought to determine the current level of resistance in Haemophilus influenzae and Streptococcus pneumoniae, the primary pathogens of pediatric conjunctivitis. Between January 1997 and March 1998, we prospectively cultured acute conjunctivitis in 250 ambulatory pediatric patients from rural Kentucky whose average age was 24.3 months. In those 250 cases, 106 H. influenzae (42% of the total) and 75 S. pneumoniae (30% of the total) pathogens were isolated, with no growth or no pathogen resulting in 79 cases (32% of the total). Beta-lactamase was detected in 60 (69%) of 87 tested strains of H. influenzae. Among 65 isolates of S. pneumoniae tested for penicillin susceptibility, 44 (68%) were susceptible, 17 (26%) were resistant, and 4 (6%) were intermediate. Conjunctivitis with acute otitis media was observed in 97 patients (39%), and H. influenzae was recovered in 57% of these 97 cases. As for in vitro activity, ciprofloxacin, ofloxacin, and tetracycline were the most active; and gentamicin, tobramycin, polymyxin B-trimethoprim, and polymyxin B-neomycin were intermediately active. Sulfamethoxazole possessed no activity against either pathogen. Beta-lactamase production was detected in 69% of H. influenzae strains, which still remains the primary causative pathogen of both conjunctivitis and conjunctivitis-otitis syndrome. Penicillin-nonsusceptible S. pneumoniae was observed in 32% of 65 patients with S. pneumoniae conjunctivitis, with most strains being penicillin resistant.