We describe a radioimmunoassay procedure for the measurement of phenytoin (5,5-diphenylhydantoin) in serum samples. Antiserum to phenytoin has been produced against phenytoin-valeric acid-bovine serum albumin conjugate.125I labelled phenytoin-acetic acid-tyrosine methyl ester has been used as a tracer. The assay covers a range of 10–500 ng/cm3 and has a sensitivity of 0.25 ng. The assay is validated by specificity tests, precision profile and recovery tests.