Various diagnostic methods have been described to detect Neisseria gonorrhoeae. Collection of first void urine is advantageous because it is non-invasive, reproducible, and painless; and provides specimens that have already been used for detection of N. gonorrhoeae by molecular tools. The purpose of this study was to assess the usefulness of first void urine for detection of N. gonorrhoeae using conventional bacteriologic techniques in patients with low-grade symptoms. Investigation was focused on first void urine and urethral secretion specimens collected from 87 male patients who were undergoing diagnotic workup for suspicion of sexually transmitted infection. Direct microscopic examination of smears stained using the Gram technique and cultures on modified Thayer-Martin medium and on cooked blood agar were performed on each specimen. The prevalence of urethritis was 58.0%. Gonorrhoea was diagnosed in 7.5% of cases. The sensitivity and specificity of microscopic examination of first void urine for detection of Gram-negative diplococci were 85.7% and 97.5% respectively. First void urine was less productive than urethral secretion for detection of urethritis: sensitivity, 44.4% and specificity, 100%, and urethral flora: sensitivity, 59% and specificity of 96.9%. The good performance of first void urine specimens for detection of Gram-negative diplococci by microscopy may justify their use for identification of N. gonorrhoeae in level 1 laboratories. First void urine could also be useful for epidemiological studies and large-scale screening surveys.