The appreciation of Ureaplasma urealyticum as a human pathogen and documentation of antibiotic resistance have heightened interest in drug susceptibilities and treatment alternatives for patients infected with this organism. Neonates pose special problems when therapy must be considered because of potential toxicities, clinical unfamiliarity or lack of experience. Forty-three isolates of U. urealyticum obtained from the lower respiratory tracts of neonates were tested against chloramphenicol, ciprofloxacin, clindamycin, erythromycin, doxycycline, and gentamicin by a microbroth dilution technique in 10B broth. In vitro resistance was observed in 1 or more strains for each of the drugs tested, except for erythromycin (minimal inhibitory concentration (MIC) range, 0.125 to 4 μg/ml, MIC 90 = 2 μg/ml). MIC90 values for the remaining five antibiotics were: doxycycline, 2 μg/ml; chloramphenicol, 8 μg/ml; ciprofloxacin, 8 μg/ml; clindamycin, 16 μg/ml; and gentamicin, 32 μg/ml. The effect of pH and/or media components on MICs was evaluated by comparing MICs of American Type Culture Collection reference strain Staphylococcus aureus 29213 obtained in Mueller-Hinton broth (pH 7.2 to 7.4) and 10B broth (pH 6.0). No appreciable effect was detected for ciprofloxacin, chloramphenicol or doxycycline, whereas gentamicin, erythromycin and clindamycin all had MICs elevated by one to several dilutions when tested in 10B broth. In some instances the difference was sufficient to alter the interpretation of the MIC. Clinical experience in treating neonatal ureaplasmal infections is reviewed along with recommendations for obtaining cultures, initiating and monitoring efficacy of therapy. © 1992 by Williams and Wilkins.