Phytochrome acts as the photoreceptor for many responses in plants, including photoperiodic control of flowering. However, the correlation between PFR and PR forms of phytochrome and flower induction have been difficult to establish, leading to the suggestion that only a small pool of the total phytochrome in photoresponsive tissues serves to monitor daylength. To investigate potential phytochrome pools in a model photoperiodic system, four distinct PHY genes were cloned and characterized from the qualitative short-day species Pharbitis nilt. PCR-based cloning was carried out using degenerate oligonucleotide primers derived from conserved regions of phytochrome genes in other higher plant species and both genomic DNA and cotyledon cDNA as templates. Sequence analysis indicated that the PCR fragments encoded PHYA, PHYB, PHYC and PHYE homologs based on comparison to Arabidopsis PHY genes. Southern blot hybridization analysis suggested that each PHY gene is represented only once in the P. nil genome and RNA blot hybridization analysis indicated that PHYA mRNA is more abundant than the other PHY mRNAs in cotyledons. The PHYA mRNA was down regulated by white and red light, PHYB and PHYC mRNA levels were not regulated by light, whereas PHYE mRNA abundance was up regulated by light. Expression of PHYA, PHYB and PHYE genes was inhibited by night break (NB) treatment but PHYC expression was not affected. Finally, the levels of PHYE mRNA abundance in cotyledons showed a pattern of regulation by a circadian rhythm over an extended 64h dark period, a treatment that resulted in floral induction. Taken together, these findings indicate that there are at least four distinct PHY genes in Pharbitis nil, and that the product of one of these genes, PHYE, may comprise a distinct phytochrome pool that specifically participates in photoperiodic timekeeping and the induction of flowering.