Usual methods allowing the measurement of the free concentration of a drug in serum, i.e. equilibrium dialysis, ultrafiltration and ultracentrifugation, are generally based on a physical separation of the bound and free fractions. During this, variations or errors may occur which are probably at the origin of the variability of the previously published results for methotrexate (CAS 59-05-2). In order to verify these results as well as to experience a technique recently applied to rifampicine the first derivative spectroscopic analysis was used to estimate the bound and free fractions of methotrexate in human serum and serum albumin (HSA). Free drug concentrations were measured at 377 nm and the bound form at 372.5 nm. In human serum, bound methotrexate was 54.1% on average for total concentrations ranging from 10(-5) mol/l to 10(-3) mol/l, without any saturation. With HSA, a saturation occurred. Scatchard analysis showed one family of binding sites characterized by 2 binding sites and an affinity constant of 3200 mol/l, in mean, values close to that previously calculated using equilibrium dialysis.