A major task in urine drug testing is to detect heroin intake. The most common way of doing this is by using morphine as the analytical target in opiate immunoassay screening. However, this strategy sometimes leads to false-positive results because morphine is not a metabolite unique to heroin. The objective of this study was to evaluate the usefulness of the unique heroin metabolite 6-acetylmorphine (6-AM) as the primary analytical target in combination with morphine in the screening assay. A total number of 3521 randomly collected urine samples from 707 patients undergoing heroin substitution treatment were investigated for 6-AM and opiates by CEDIA (cloned enzyme donor immunoassay) and for opiates by DRI immunoassays and by gas chromatography-mass spectrometry (free 6-AM, free morphine, total morphine, and total codeine). The rate of positive outcome in the screening for 6-AM was 9.1% (cutoff 10 microg/L), and for opiates, it was 22.6% (cutoff 300 microg/L), which is in accordance with a known shorter detection time for 6-AM following heroin intake. However, by comparing 6-AM and opiate screening results at different cutoff levels, it was observed that 7-8% of the samples and 12.5% of the patients with detectable 6-AM had an unexpected low content of free and total morphine in the urine. This study confirms earlier observations that certain individuals may escape detection in urine drug testing when morphine is being utilized for the detection of heroin intake. The underlying mechanism for this may be a metabolic defect and/or interaction. It is concluded that 6-AM is a valuable target analyte in the screening of drugs of abuse in urine and may be used in combination with opiate screening in clinical testing.