The protein binding of methotrexate by serum from eight normal volunteers was assessed by continuous ultrafiltration at pH 7.4 and 37 degrees C. Methotrexate concentrations were measured by radioimmunoassay and the data analysed by the method of Scatchard. The major binding protein was albumin which bound 87.3% of the drug in serum. Analysis of the Scatchard plots indicated two distinct groups of binding sites. Class I was found to have 0.16 +/- 0.05 (S D) binding sites with an intrinsic association constant of 71.15 +/- 35.98 (S D) X 10(4) M-1: Class II had 2.01 +/- 0.93 (S D) binding sites and and affinity of 0.18 +/- 0.15 X 10(4) M-1. No great change in the percentage of methotrexate bound occurred until the total concentration of the drug exceeded 50 muMol 1-1.