A nuclear gene, sigA, that encodes a sigma factor for chloroplast RNA polymerase has previously been identified and characterized in the primitive red alga Cyanidium caldarium strain RK-1. Southern hybridization analysis indicated the presence of two additional sigma factor genes, which have now been cloned and shown to encode virtually identical proteins that are homologous to eubacterial sigma factors. These genes, which are also present in the nuclear genome, have therefore been named sigB and sigC. The substantial sequence similarity of sigB and sigC to sigA of the same strain as well as to cyanobacterial principal sigma factors and other chloroplast sigma factors strongly suggests that the nuclear genome of C. caldarium contains three genes that encode two types of chloroplast sigma factors. Each of the three recombinant Sig proteins showed sigma factor activity in vitro when combined with the Escherichia coli RNA polymerase core enzyme. Northern blot analysis revealed that, whereas the overall abundance of sigA transcripts was not affected by light, the amount of sigB and sigC mRNAs was greater in the light than in the dark. Thus, multiple sigma factors appear to contribute to light-regulated gene expression in the chloroplast.