18 strains of thymidine-requiring streptococcal mutants (thy-) were isolated from urines of patients with urinary tract infection treated with trimethoprim-sulfamethoxazole (TMP-SMZ) or trimethoprim-sulfadiazine (TMP-SDZ). All thy- mutants were catalase- and β-lactamase-negative, grew in the presence of bile and esculin, and required from 0.3 to > 1280 μg/ml of thymidine for normal growth. The antibiotic susceptibility of wild-type and thy- mutants to 21 antimicrobial agents tested were comparable except to trimethoprim (TMP), TMP-SMZ and TMP-SDZ. Ampicillin, penicillin G, erythromycin and rifampin were among the most active compounds tested. Growth kinetic studies with a Streptococcus faecalis thy- mutant in a synthetic basal medium without thymidine resulted in a decrease of 2–3 logarithmic units in viable cells after 24 h of incubation. The addition of thymidine to this thymidine-deprived culture prevented the thymineless death of the cells. In vivo, this thy- mutant was less virulent than the wild-type strain in producing kidney infection in mice.