All known DNA tumor viruses are known to target and inactivate two main cell cycle regulatory proteins, retinoblastoma protein (pRb) and p53. Inactivation of pRb promotes host cell cycle progression into S phase, and inactivation of p53 promotes cell immortalization. The DNA tumor virus Kaposi's sarcoma associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) was shown to target and inactivate pRb as well as p53. In this report we provide evidence that these functions are conserved in the homologous protein encoded by the related gammaherpesvirus herpesvirus saimiri (HVS). ORF73, the HVS homologue of LANA, is shown to bind both p53 and pRb in vitro and in vivo, to colocalize with p53 in human T cells infected with HVS, and in cells overexpressing both ORF73 and p53, as well as to adversely influence pRB/E2F and p53 transcriptional regulation. The C terminus of LANA, the region most highly conserved in ORF73, is shown to be responsible for both pRb and p53 interactions, supporting the hypothesis that these functions are conserved in both homologues. Finally, the region of p53 targeted by LANA (and ORF73) maps to the domain required for tetramerization. However, preliminary cross-linking studies do not detect disruption of p53 tetramerization by either LANA or HVS-encoded ORF73, suggesting that p53 inactivation may be by a mechanism independent of tetramer disruption.