Detection of Chlamydia trachomatis and/or Neisseria gonorrhoeae infection in urine using molecular amplification assays has permitted institutions with limited medical facilities to offer testing for these sexually transmitted diseases (STDs). The Nebraska Public Health Laboratory (NPHL) investigated the validity of urine samples submitted for C trachomatis and/or N gonorrhoeae amplification after receiving a substantial number of clear specimens. Approximately 75% of all urine specimens submitted for STD testing to the NPHL were from correctional facilities. The falsification of urine specimens submitted for microbiology studies is not evaluated routinely, and this problem was previously undocumented. By using the criteria for specific gravity of 1.001 or less and a creatinine concentration of less than 5 mg/dL (442 mumol/L), approximately 8% of all specimens submitted during the study interval were determined to be inconsistent with urine. The microbiology laboratory should be aware of the possibility for specimen manipulation to identify facilities submitting falsified specimens, to initiate appropriate intervention, and to minimize false-negative reporting.