We obtained specimens for culture from the lids and conjunctivae of 95 patients with acute conjunctivitis and 91 control children of similar age and, in addition, stained the conjunctival scrapings with Giemsa and Gram stains. The conjunctivitis was attributed to bacterial infection in 76 patients, viral infection in 12 children, and allergy in 2 patients; no cause was identified in the remaining 5 patients. In most cases the etiologic diagnosis was based on the results of laboratory studies. By separately culturing microorganisms in specimens from the lids and conjunctivae of patients and control subjects, we could distinguish normal flora from pathogens, and blepharitis from conjunctivitis. Staphylococci, corynebacteria, and alpha-hemolytic streptococci were the predominant organisms recovered from the lids of control subjects. In contrast, Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis were the major pathogens cultured from the conjunctival specimens from patients with bacterial conjunctivitis. Gram stains of conjunctival scrapings provided a rapid means of predicting the pathogen in 51 of 55 cases of bacterial conjunctivitis. Giemsa stains of conjunctival scrapings provided etiologic information in 81 of 84 cases, showing neutrophilia in bacterial infections, lymphocytosis in viral infections, and eosinophilia in allergic disease. These results indicate that most cases of acute conjunctivitis in children can be diagnosed on the basis of differential cultures of microorganisms from the lid and conjunctiva, together with Giemsa stains of conjunctival scrapings.