We assayed Type 1 (high K+) and Type 2 (high Na+) human breast cyst fluids for DHEA-S. When an antibody specific for the 3-sulfoconjugate end of DHEA-S was used, Type 1 cyst fluids (n = 18) showed a content of 114 +/- 68 micrograms/mL (mean +/- sigma) and Type 2 cyst fluids (n = 14) of 35 +/- 17 micrograms/mL (P less than 0.01). Using an antibody specific for the D-ring, the results were 151 +/- 91 micrograms/mL and 51 +/- 32 micrograms/mL, respectively (P less than 0.01). The apparent concentrations of DHEA-S were statistically different, even though both assays gave equal results in serum from normal adults. The presence of other compounds in individual cyst fluid samples was examined by extraction and chromatography. DHEA-S immunoreactivity was found in both early and late eluting fractions in Type 1 cyst fluids and in late eluting fractions from Type 2 cyst fluids. Only the late eluting fraction from Type 2 fluids had approximately equal immunoreactivity with both antibodies. In addition to authentic DHEA-S, breast cyst fluids contain other materials that react with DHEA-S antibodies. Radioimmunoassays for DHEA-S in cyst fluid must be specifically validated because of the presence of these compounds.