The in vitro activities of two investigational quinolones, sparfloxacin (previously designated AT 4140) and PD 127391, were determined for 30 strains each of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum and compared with those of ciprofloxacin, tetracycline, clindamycin, and erythromycin. Erythromycin was the most active compound against M. pneumoniae (maximum MIC, <0.008 μg/ml). PD 127391 (MICs, <0.008 to 0.031 μg/ml), sparfloxacin (MICs, <0.008 to 0.25 μg/ml), clindamycin (MICs, <0.008 to 0.5 μg/ml), and tetracycline (MICs, 0.063 to 0.25 μg/ml) were superior to ciprofloxacin (MICs, 0.5 to 2 μg/ml). Sparfloxacin and PD 127391 were active against M. hominis (MICs, <0.008 to 0.031 μg/ml for each) at concentrations comparable to those of clindamycin (MICs, <0.008 to 0.063 μg/ml) and at concentrations lower than those of ciprofloxacin (MICs, 0.125 to 0.5 μg/ml). As expected, M. hominis was resistant to erythromycin (MICs, 32 to ≥256 μg/ml). For U. urealyticum, PD 127391 (MICs, 0.031 to 0.5 μg/ml) and sparfloxacin (MICs, 0.063 to 1 μg/ml) were superior to erythromycin (MICs, 0.25 to 4 μg/ml), ciprofloxacin (MICs, 0.5 to 8 μg/ml), and clindamycin (MICs, 0.25 to 64 μg/ml. Both new quinolones were equally active against tetracycline-susceptible as well as -resistant strains of M. hominis and U. urealyticum. The possible influence of medium components and/or pH on MICs was evaluated by testing a Staphylococcus aureus reference strain with each antibiotic in SP-4 broth and 10-B broth and comparing the results with published MICs for this strain. MICs determined in 10-B broth for erythromycin were affected most. This study shows that the activities of sparfloxacin and PD 127391 are similar to one another and comparable or superior to those of other drugs used to treat mycoplasmal infections. The MICs of both new quinolones were consistently 2 to several dilutions lower than those of ciprofloxacin for each species.