Two homologs of the p53 tumor suppressor, p63 and p73 have recently been discovered. These proteins have activities similar to p53 in cell culture but have distinct developmental functions in vivo. We found that temperature-sensitive mutants of certain p63 and p73 isoforms can be created by single amino acid substitutions of an alanine residue corresponding to alanine 135 of murine p53. The mutants (p63 gamma -Pro167, p73 alpha -Leu156 and p73 beta -Ile156) can be controlled by temperature shift between 32 degreesC and 39 degreesC. They can be stably expressed in p53-null H1299 cells at 39 degreesC, become transcriptionally activated at 32 degreesC, and induce expression of p53-responsive genes MDM2 and p21WAF1. Activation of p73 beta -Ile156 in H1299 cells inhibits cell division but induces significant increase in cell size (hypertrophy), whereas activation of p73 alpha -Leu156 and p63y-Pro167 induces apoptosis. These mutants may be useful tools for gaining further insight to the functions of p53 homologs.