We used simulated blood cultures inoculated with clinical isolates of Mycoplasma hominis to determine whether liquid media of the BacT/ALERT (Organon Teknika, Durham, N.C.) will support growth of this fastidious organism and whether its presence can generate a positive signal with the instrument. Viability of clinical isolates of M. hominis was maintained for 7 days in BacT/ALERT media, and organisms were able to multiply when 1% gelatin was added to neutralize the mycoplasmastatic effects of the sodium polyanetholsul fonate anticoagulant. Without the addition of gelatin to BacT/ALERT bottles, the mycoplasmas declined in numbers or became completely nonviable. Mycoplasmal growth was further enhanced in BacT/ALERT PF both supplemented with gelatin, arginine, and DNA in comparison to broth with only gelatin added. No BacT/ ALERT bottles containing M. hominis in simulated blood cultures were flagged positive by the instrument, despite growth of microorganisms of up to 107 CFU/ml after incubation for up to 7 days, suggesting that inadequate CO2 production or some other mechanism prevents the instrument from recognizing the presence of the organism and its metabolic products. The fastidious cultivation requirements and relatively slow growth of M. hominis warrant that dependence on automated systems and techniques designed to detect conventional bacteria will not be reliable for recovery of M. hominis and that specialized media and incubation conditions designed for optimum cultivation of mycoplasmas should be employed when this organism is suspected on clinical grounds.