The p53 family of proteins contains two members that have been implicated in sensitization of cells and organisms to genotoxic stress, i.e., p53 itself and p73. In vitro, lack of either p53 or p73 can protect certain cell types in the adult organism against death upon exposure to DNA damaging agents. The present study was designed to assess the relative contribution of p53 to radiation resistance of an emerging vertebrate model organism, i.e., zebrafish embryos. Consistent with previous reports, suppressing p53 protein expression using antisense morpholino oligonucleotides (MOs) increased survival and reduced gross morphological alterations in zebrafish embryos exposed to ionizing radiation. By contrast, a pharmacological inhibitor of p53 function [Pifithrin-alpha(PFTalpha)] caused developmental abnormalities affecting the head, brain, eyes and kidney function and did not protect against lethal effects of ionizing radiation when administered at 3 hours post fertilization (hpf). The phenotypic abnormalities associated with PFTalpha treatment were similar to those caused by antisense MO knock down (kd) used to reduce p73 expression. PFTalpha also inhibited p73-dependent transcription of a reporter gene construct containing canonical p53-responsive promoter sequences. Notably, when administered at later stages of development (23 hpf), PFTalpha did not cause overt developmental defects but exerted radioprotective effects in zebrafish embryos. In summary, this study highlights off-target effects of the pharmacological p53 inhibitor PFTalpha related to inhibition of p73 function and essential roles of p73 at early but not later stages of zebrafish development.