The addition of polyethyleneglycol (PEG) side chains to interferon alpha-2a improves the serum stability and clinical efficacy. Current commercial PEG-INF formulations such as PEGASYS are heterogeneous and contain multiple monopegylated isomers. We have analyzed the activity of nine, purified monopegylated variants in antiproliferative, antiviral and binding assays, together with a global transcriptional analysis using DNA oligonucleotide microarrays. We show a direct correlation between biological and transcriptional activity for all isomers and an inversed correlation between IFN-receptor 2a affinity and signal transduction. Two out of nine positional isomers have a higher specific biological and transcriptional activity than the mixture, which can be explained by unique structural features of interferon signaling, which involves two distinct receptors. The possible clinical implications are discussed, which might guide the development of pegylated interferons with improved pharmacological properties.