Lid isolates of Staphylococcus aureus and coagulase-negative staphylococci (CNS) from controls (12 S. aureus and 110 CNS) and from patients with blepharitis (17 S. aureus and 171 CNS) were tested for production of alpha, beta, delta, epsilon, and previously undescribed hemolytic toxins, because toxin production has been implicated as a cause of blepharoconjunctivitis. The electrolyte content of agar media required for toxin production was first investigated. Alpha-lysin was found to be produced by all isolates of S. aureus colonizing lids of normal controls and patients with blepharitis, but by none of 281 CNS isolates. A new toxin was identified, having low molecular weight (5 kd), produced by one CNS strain isolated from a blepharitic lid. It was produced on basic nutrient agar that lacked sodium but contained glucose, which inhibited production of alpha-lysin. It hemolyzed rabbit and sheep erythrocytes and, surprisingly, was neutralized by polyclonal antiserum to alpha-lysin. This may explain occasional reports of alpha-lysin production by CNS. The overall results do not support a hypothesis of hemolytic toxin production by staphylococci as a general cause of blepharitis.