The Roche Abuscreen Onlinetrade mark Amphetamine immunoassay (IA), modified to include sodium periodate, and the Microgenics DRI Ecstasy IA were used to determine the prevalence of amphetamine (AMP), methamphetamine (MAMP), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA) in urine specimens from applicants seeking to join the United States Armed Forces. Over a 4-month period, a total of 85,658 specimens were IA screened using the Department of Defense 500 ng/mL administrative cutoff level for AMP and MDMA. All presumptively positive specimens were confirmed using a solid-phase extraction procedure coupled with simultaneous analysis of AMP, MAMP, MDA, MDMA, and MDEA by fast gas chromatography-mass spectrometry using the same cutoff levels as the IA. The Roche Online Amphetamine IA identified 216 specimens as presumptively positive; of these, 70 specimens confirmed positive for AMP and 87 specimens confirmed positive for AMP and/or MAMP, resulting in a confirmation rate of 73%. The Microgenics DRI Ecstasy IA identified eight specimens as presumptively positive; of these, five specimens confirmed positive for MDMA and/or MDA, resulting in a confirmation rate of 63%. The total use prevalence for AMP, MAMP, MDA, MDMA, and/or MDEA in military entrance processing stations specimens over the testing period was determined to be 0.19%.