Spironolactone and one of its metabolites, canrenone, cross-react with some digoxin immunoassays to result in erroneous serum digoxin concentrations. Recently, additional compounds, 7-alpha-thiomethylspirolactone (7-a-TMS) and 6-beta-hydroxy-7-alpha-thiomethylspirolactone (6-B-OH-7-a-TMS), have been reported to be quantitatively important metabolites of spironolactone. This study was initiated to evaluate the cross-reactivity of these metabolites, canrenone, and spironolactone with four different digoxin immunoassays. Blank serum was spiked with each compound to yield concentrations reported to occur in vivo. Samples were analyzed in duplicate by each of the following immunoassays: fluorescence polarization immunoassay (FPIA); affinity-column-mediated immunoassay (ACMIA); radioimmunoassay (RIA); and enzyme immunoassay (EIA). The 7-a-TMS metabolite cross-reacted with both the RIA and ACMIA methods. Apparent digoxin concentrations were as great as 0.39 ng/ml for this metabolite at the highest concentration evaluated, 600 ng/ml. At the lowest concentrations evaluated with the 7-a-TMS metabolite, 50 ng/ml, apparent digoxin concentrations as high as 0.28 ng/ml were reported. The 6-B-OH-7-a-TMS metabolite did not cross-react to a significant extent with any of immunoassays studied. Canrenone cross-reacted with the ACMIA method at a concentration of 100 ng/ml. The EIA method exhibited no apparent cross-reactivity with any of the compounds, whereas the FPIA method exhibited minimal cross-reactivity. The results of this study indicate that the 7-a-TMS metabolite cross-reacts to a significant extent with some immunoassays; however, this is not true for the 6-B-OH-7-a-TMS metabolite.