This report describes the effect of flavin adenine dinucleotide (FAD) and its precursor vitamin B2on an apoenzyme reactivation immunoassay system (ARIS) both in vitro and in vivo. It was observed that 10 nM/L FAD in the buffer causes a false-positive result in ARIS for theophylline by the value of 94 mUM/L, whereas vitamin B2, flavin mononucleotide, adenosine, adenosine monophosphate, and adenosine diphosphate (ADP) at the same concentration caused no such effect. Nevertheless, ADP at 2 mUM/L can cause a drop in theophylline reading of 55 mUM/L. However, the sera from four volunteers with normal liver functions who had taken 30 mg of vitamin B2daily for a week showed no effect on theophylline reading.