Four natural murine interferon-alpha genes (MuIFN-alpha 1, -alpha 2, -alpha 4 and -alpha 6) and four hybrid genes (alpha 1 alpha 4, alpha 2 alpha 4, alpha 4 alpha 1 and alpha 4 alpha 2) were transiently expressed in monkey COS cells under the transcriptional control of the simian virus 40 early promoter. The proteins were labelled with [35S]methionine during a 16 h incubation and proteins secreted by the cells during this period were separated by polyacrylamide gel electrophoresis and subsequently visualized by fluorography. Under the conditions used, the IFNs represented 5 to 10% of the total amount of secreted proteins. All genes were found to encode biologically active IFN subspecies, including alpha 4 which has a deletion of five amino acids. When the specific activities of the proteins were compared, it appeared that the specific antiviral activity of alpha 4 on mouse cells was three- to sixfold higher than the activities of the other natural IFN subspecies. The specific activities of the hybrid proteins were similar to those of the natural proteins, except for the alpha 2 alpha 4 hybrid which had a higher specific activity than the original proteins. The ability of the natural and hybrid subspecies to protect hamster cells against viral infection was determined using MuIFN-alpha 1 as a standard. Large differences in activity were found, with alpha 6 as the most and alpha 4 as the least active subspecies.