Type III interferon (IFN) or IFN-lambda is a recently discovered family of IFNs that signal through the same downstream transcription factors as type I IFN but use a separate receptor complex composed of the IL-10R2 and the unique IFN-lambdaR1 receptor chains. We have established a simple and efficient expression system to produce highly pure and active IFN-lambda of the three human IFN-lambda isoforms (IFN-lambda1, -lambda2 and -lambda3) and used this to compare the biological activity of the different IFN-lambda subtypes. Surprisingly, we found IFN-lambda3 to possess the highest specific activity of the human IFN-lambda subtypes, exhibiting a twofold higher activity than IFN-lambda1 and a 16-fold higher activity than IFN-lambda2. Furthermore, in comparison with the commercially available preparations of IFN-lambda1 and -lambda2, we found our IFN-lambda preparation to be superior in activity.