Recent evidence has suggested that surface structures of pathogenic bacteria, which are important in attachment to human mucosal surfaces, may be absent on bacteria grown in the presence of subinhibitory concentrations of antibiotics. We studied the effect of tetracycline and penicillin on meningococcal and gonococcal pili. Subinhibitory concentrations of tetracycline and penicillin were found to markedly reduce the number of pili per meningococcus or gonococcus and the percentage of meningococci or gonococci with pili, as determined by negative-staining electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane preparations suggested that tetracycline decreased expression of pili by inhibiting synthesis of pilin subunits. In contrast, pilin subunit synthesis was unaltered by penicillin, suggesting a defect in assembly of pilin subunits or in anchoring of assembled pili. The decrease in the number of pili that occurred with subinhibitory concentrations of both tetracycline and penicillin was accompanied by a marked decrease in the ability of the organisms to attach to human cells. Gonococci or meningococci removed from the influence of subinhibitory concentrations of the antibiotics regained piliation, and attachment returned to levels near those of controls. The expression of meningococcal and gonococcal pili may be affected by factors that influence synthesis of pilin subunits or factors that interfere with the assembly and anchoring of pili in the outer membrane.