Objective: Until recently the generally accepted paradigm implied that urine of healthy people is sterile. In the present study, urine of healthy subjects was investigated by extended bacteriological methods. Material and Methods: Three midstream urine samples from 52 healthy subjects each (24 females, 28 males; 18-25 years of age) were investigated by an extended set of culture media for identification of facultative aerobic (FAB) and nonclostridial anaerobic bacteria (NCAB). Ward's method (Euclidean distance) was used for similarity analysis. Results: The bacterial count of FAB in urine was usually low (≤102 colony-forming units/ml) in both groups. In contrast, the bacterial count of NCAB was higher (≥103 colony-forming units/ml), at least in some species, with significant differences between genders. The average number of bacterial species found was 5.8 in female and 7.1 in male urine. Half of the females were assigned to a specific ‘female' microbial spectrum, different from that of males. In the mixed-gender clusters, the males showed a greater similarity among themselves. Conclusions: As also shown by other investigators, urine of healthy people is normally not sterile. The role of the routinely not cultivated bacteria in healthy and diseased subjects needs to be established. It may alter the diagnostics of infectious and inflammatory diseases of the urogenital tract.