The murine double-stranded RNA-binding protein RAX and the human homolog PACT were originally characterized as activators of protein kinase R (PKR). Mice deficient in RAX show reproductive and developmental defects, including reduced body size, craniofacial defects and anterior pituitary hypoplasia. As these defects are not observed in PKR-deficient mice, the phenotype has been attributed to PKR-independent activities of RAX. Here we further investigated the involvement of PKR in the physiological function of RAX, by generating rax-/- mice deficient in PKR, or carrying a kinase-inactive mutant of PKR (K271R) or an unphosphorylatable mutant of the PKR substrate eIF2alpha (S51A). Ablating PKR expression rescued the developmental and reproductive deficiencies in rax-/- mice. Generating rax-/- mice with a kinase-inactive mutant of PKR resulted in similar rescue, confirming that the rax-/- defects are PKR dependent; specifically that the kinase activity of PKR was required for these defects. Moreover, generating rax-/- mice that were heterozygous for an unphosphorylatable mutant eIF2alpha provides partial rescue of the rax-/- defect, consistent with mutation of one copy of the Eif2s1 gene. These observations were further investigated in vitro by reducing RAX expression in anterior pituitary cells, resulting in increased PKR activity and induction of the PKR-regulated cyclin-dependent kinase inhibitor p21WAF1/CIP1 . These results demonstrate that PKR kinase activity is required for onset of the rax-/- phenotype, implying an unexpected function for RAX as a negative regulator of PKR in the context of postnatal anterior pituitary tissue, and identify a critical role for the regulation of PKR activity for normal development. This article is protected by copyright. All rights reserved.