The protein kinase inhibitor (PKI) family includes three genes encoding small, heat-stable inhibitors of the cyclic AMP-dependent kinase PKA. Each PKI isoform contains a PKA inhibitory domain and a nuclear export domain, enabling PKI to both inhibit PKA and remove it from the nucleus. The PKIbeta isoform, also known as testis PKI, is highly expressed in germ cells of the testis and is found at more modest levels in other tissues. In order to investigate its physiological role, we have generated PKIbeta knockout mice by gene targeting. These mice exhibit a partial loss of PKI activity in testis but remain fertile with normal testis development and function. PKIbeta knockout females also reproduce normally. The PKIbeta mutants were crossed with our previously derived PKIalpha mutants to obtain double-knockout mice. Remarkably, these mice are also viable and fertile with no obvious physiological defects in either males or females.