One or more digoxin-like immunoreactive substances (DLIS), most frequently present in serum of premature and full-term neonates. cross react to various extents with different digoxin immunoassay kit reagents. Mostly, this variation is attributed to the relative cross reactivity of DLIS with the antiserum in each kit. However, modification of standard assay procedures for digoxin can also greatly alter the relative cross reactivity of DLIS. Using sequential RIA kit methods for digoxin by 20 to 60% relative to the standard equilibrium RIA mode. Cross reactivity was decreased still more if the concentrations of antiserum (binding-site concentration) and tracer (125 l-labeled digoxin) were decreased, and conversely. Serum samples containing only digoxin, analyzed by the modified method, consistently yielded results well comparable with those obtained with the manufacturers' recommended procedures. We describe use of the different responses to digoxin and DLIS of standard and sequential radioimmunoassays and use of simultaneous equation to calculate the concentration of DLIS (in digoxin equivalents) in digoxin-containing samples.