The binding of thyroxine to prealbumin in normal human serum has been quantitated by electrophoresis in agarose gels made up in 0.02m sodium phosphate buffer. Thyroxine binding to prealbumin is dependent on pH. For 9 individual sera studied at 37 C and pH 7.4, prealbumin bound a mean of 13.7±1.1% (sd) of the tracer thyroxine-131I, while at pH 8.6 mean binding was 25.2±3.5% (sd), 1.8 times more than at pH 7.4. This effect of pH was seen with every serum sample. Significantly less thyroxine was bound to prealbumin at pH 7.0 than at 7.4 and slightly less at pH 9.0 than at 8.6. Only a small effect of temperature was seen. At 31 C and pH 7.4, prealbumin binding was about 30% greater than at 37 C, but at 42 C binding was not significantly different from that at 37 C. These results, in which some 85% of thyroxine is bound to thyroxine-binding globulin plus albumin and only about 15% to prealbumin at pH 7.4 and 37 C, suggest that prealbumin, contrary to recently held views, is only a minor carrier of thyroxine in human plasma under physiologic circumstances.