The Epstein-Barr virus (EBV) EBER transcripts are small, highly structured RNAs able to bind to and inhibit activation of the double-stranded RNA-dependent protein kinase PKR in cell-free systems, and within latently infected B-cell lines they inhibit alpha interferon-induced apoptosis that is believed to be mediated through PKR. Here, we address the consequences of EBER expression for PKR activation in vivo in response to alpha interferon. In agreement with published findings, either EBV infection or the EBERs alone protected Burkitt lymphoma cells from alpha-interferon-induced apoptosis. However, utilizing multiple phosphorylation state-specific antibodies to monitor PKR activation within cells in response to interferon, we demonstrate that the EBERs are unable to inhibit phosphorylation of either cytoplasmic or nuclear PKR. Concordantly, a direct substrate of PKR, the ␣ subunit of eukaryotic initiation factor 2 (eIF-2␣), was equally phosphorylated in EBV-positive and EBV-negative cells following interferon treatment. Therefore, EBER inhibition of alphainterferon-induced apoptosis, and potentially other PKR-mediated events, is unlikely to be mediated through direct inhibition of PKR, as previously thought.