Melatonin was extracted from serum by using Baker reversed-phase C-18 columns. More than 99% of the applied melatonin was retained by the columns, and more than 97% was eluted from the columns in 300 microL of methanol. We then determined melatonin in the serum extract by a modification of a standard radioimmunoassay, using filtration instead of centrifugation to collect the [3H]melatonin-antibody complex precipitated by saturated ammonium sulfate. These modifications allow more rapid, accurate, and reproducible determination of melatonin than do previously published procedures.