A latex-agglutination inhibition test was developed and evaluated for the detection of barbiturates, primarily in urine, but it has applicability to serum. The 2-h test was performed with samples free of gross debris in a 37 degree C heat block and the reaction between the barbiturate antibody and the barbiturate-latex, at pH 7.3, was inhibited by urines containing secobarbital, amobarbital, pentobarbital, butabarbital, or phenobarbital. For laboratory standards the test was particularly sensitive to secobarbital (300 microgram/litre) and relatively insensitive to amobarbital (5000 microgram/litre). In clinical specimens some barbiturates were detected at levels as low as 150 microgram/litre. The test was specific, and negative endpoints were frequently noted in as little as 30 min. The effects of variables (antiserum dilution, urine specific gravity, heat block temperature, sample size, and type of barbiturate) on the degree of agglutination were also determined.