The pleiotropic biologic effects of interferon (IFN) are mediated through regulation of the expression of numerous IFN-sensitive genes. Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors were analyzed to study the immunoregulatory and antiviral messenger RNAs (mRNAs) and proteins regulated by pegylated IFN-α2b (PEG-IFN-α2b) and IFN-α2b. A dose-dependent and time-dependent response for multiple IFN-regulated genes was observed. IFN-dependent protein production and secretion were correlated with IFN-regulated mRNA induction. Overall regulation of gene expression patterns for PEG-IFN-α2b and IFN-α2b was comparable, even though the antiviral activity of PEG-IFN-α2b demonstrated a longer biologic halflife in vitro compared with IFN-α2b. To study the heterogeneity of responses, PBMCs obtained from over 25 healthy donors were analyzed. Within a particular donor dataset, gene-specific and dose-dependent responses to PEG-IFN-α2b treatment, demonstrated in both the amplitude of transcriptional upregulation and the duration of sustained mRNA upregulation, were observed. However because of donor heterogeneity, the amplitude of a given transcriptional response could not be predicted for a specific dose of PEG-IFN-α2b. Notably, mRNA levels of oligoadenylate synthetase (OAS), double-stranded RNA (dsRNA)-activated protein kinase (PKR), IP-10, IFN-stimulated gene 54 (ISG54), and ISG15 were upregulated after 120 h of continuous PEG-IFN-α2b treatment. These results suggest that the use of antiviral and immunoregulatory protein mRNA levels as markers to assess the therapeutic efficacy of IFN-α2b and PEG-IFN-α2b against viral and neoplastic diseases in clinical trials is promising but will require further analysis using clinical patient samples.