Enzyme-linked ligand-sorbent assay (ELLSA) of riboflavin was performed in standard, multi-well microtitre plates. 3-Carboxymethylriboflavin was carbodiimide-coupled to bovine serum albumin and the conjugate was adsorbed on the well surface. Riboflavin-binding protein from egg-white was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester. The assay was based on competition of riboflavin analyte with the immobilized flavin for the biotinylated binder. Secondary adsorption of the biotinylated riboflavin-binding protein was measured by using avidin-bearing horseradish peroxidase label. The optimized method had a detection limit of 0.8 pmol of riboflavin and was expected to work within a riboflavin concentration range of 2 X 10(-8)(-4) X 10(-6) mol l-1. Preliminary trials suggested that ELLSA was suitable for determining riboflavin in human urine and the sum of riboflavin and flavin nucleotides in human plasma. The analytical performance of ELLSA for those materials was characterized by good consistency of the results with those obtained by conventional, fluorimetric methods, a mean recovery of riboflavin supplement of over 90% and a within-plate relative standard deviation below 20%. Some unique samples of both urine and plasma were assayed with between-plate relative standard deviations higher than 30%, implicating further modification of this version of ELLSA. The method intended for routine control determinations of vitamin B2 status in human subjects and is addressed to laboratories that routinely perform automated, microplate-based enzyme-linked assays.