We have isolated and studied the cloned sigma factors SASIG1-3 from mustard (Sinapis alba). In functional analyses using both promoter and factor mutants, the three recombinant proteins all had similar basic properties but also revealed differences in promoter preference and requirements for single nucleotide positions. Directed muta- genesis of SASIG1 identified critical residues within the conserved regions 2.4 and 4.2 necessary for binding of the −10 and −35 promoter elements, respectively. SASIG1 and 2, but not SASIG3, each have a typical region 2.5 for binding of the extended −10 promoter element. SASIG3 has a pro-sequence reminiscent of σK from Bacillus subtilis, suggesting that proteolytic cleavage from an inactive precursor is involved in the regulation of plastid transcription. In addition, SASIG2 was found to be more abundant in light-grown as compared to dark-grown mustard seedlings, while the converse was true for SASIG3.