We prepared 125I-labeled cyclosporine (125I-CS) by modifying the procedure of Mahoney and Orf (Clin Chem 1985;31:459) and characterized it with regards to maximal immunoreactivity (greater than 90%), trichloroacetic acid precipitability (greater than 90%), and stability (90% immunoreactive after five half-lives of 125I). For a particular preparation of 125I-CS, we estimated its immunoreaction concentration (50 pmol/L) and the equilibrium constant for its reaction with Sandoz polyclonal antiserum (K = 3.9 X 10(9) L/mol). By substituting 125I-CS as tracer in the Sandoz radioimmunoassay and by modifying other aspects of the assay, we developed a procedure that is sufficiently sensitive (0.34 micrograms/L) to allow measurement of "trough" (lowest inter-dose) cyclosporine concentrations in parotid saliva. Of 38 kidney-transplant patients, 35 had measurable concentrations in saliva (mean 8.3, SD 5.2 micrograms/L), and these correlated moderately with paired serum concentrations (r = 0.68, P less than 0.001). We believe that measurement of salivary cyclosporine may offer a simple way of estimating the free fraction of the drug in serum or plasma.