Photoperiodic control of flowering is regulated by light and a circadian clock. Feedback regulation of the transcription of clock components is one of the most common and important mechanisms that control clock functions in animals, fungi, and plants. The Arabidopsis circadian clock is believed to involve two myb-related proteins, LHY (late elongated hypocotyl) and CCA1 (circadian clock associated 1), which negatively regulate TOC1 (timing of cab expression 1) gene expression through direct binding to the TOC1 promoter. PIF3 (phytochrome-interacting factor 3), a bHLH transcription factor binds promoter regions of the LHY and CCA1 genes, affecting the light induction of these genes, and interacts with TOC1 protein. Although the positive feedback regulation of clock components in plants has been predicted, and PIF3 has been assumed to be involved, the molecular nature of this process has not been elucidated. Here we demonstrate that the antisense suppression of the PIF3 gene causes higher levels of mRNA of floral activator genes CO (constans) and FT (flowering locus T) and results in early flowering under long days (LD). Neither the circadian rhythms of the clock-controlled genes (CCGs) under constant conditions nor the diurnal rhythms of the CCGs under LD conditions are affected by the reduction in PIF3 gene expression. These results suggest that PIF3 may play an important role in the control of flowering through clock-independent regulation of CO and FT gene expression in Arabidopsis.