We have created two mutants of mouse transcription factor c-Rel (c-G29E and c-R266H) that are analogous to mutants previously shown to have temperature-sensitive (ts) functions for the homologous Drosophila protein Dorsal and the retroviral oncoprotein v-Rel. In vitro, c-R266H shows both a ts and a concentration-dependent ability to bind DNA, suggesting that the lesion affects the ability of c-Rel to form homodimers. In contrast, the ability of mouse c-G29E to bind DNA in vitro is not ts. c-Rel mutant c-R266H also shows a ts ability to activate transcription from a kappaB-site reporter plasmid, whereas c-G29E activates transcription well above control levels at both 33 and 39 degrees C. Insertion of two amino acids (Pro-Trp) between amino acids 266 and 267 in mouse c-Rel (mutant c-SPW) also creates a c-Rel protein with distinct properties: mutant c-SPW is partially defective in that it cannot form DNA-binding homodimers but can form DNA-binding heterodimers with p50. Interestingly, the mutations in c-Rel that affect homodimer formation (c-R266H and c-SPW) fall within a consensus protein kinase A recognition sequence but are not predicted to lie in the dimer interface. Conditional and partially defective mutants such as those described herein may be useful for identifying physiological responses and genes regulated by specific Rel/NF-kappaB family members.