Methods for determining the specific activity and percentage purity of highly purified interferon preparations are unreliable, mainly because of the difficulty in estimating very small quantities of protein. Human leukocyte-derived interferon (HuIFN-alpha) is a mixture of several distinct species, and the relationship between specific activity and purity is not direct. A non-biological method is described here, by which the purity of highly purified HuIFN-alpha is determined chromatographically. Samples are run on a Sephadex G-75 column with continuous measurement of E206 in the eluate, and SDS gel electrophoresis is used to separate essentially all the protein species in the characteristic double peak region containing interferon activity and show that they are interferons. The validity of the method has been checked using a range of standard proteins. The specific activity of pure Namalwa (lymphoblastoid) HuIFN-alpha was estimated to be 1.0--1.7 x 10(8) U/mg protein.