We isolated and characterized AtC401, a novel Arabidopsis clock-controlled gene that encodes a protein containing the pentatricopeptide repeat (PPR) motif. AtC401 was isolated as an Arabidopsis homolog of Pharbitis nil C401 (PnC401), a gene that encodes a leaf protein closely related to the photoperiodic induction of flowering and displays a circadian rhythm at the transcriptional level. The AtC401 gene spans 5.6 kb and contains 12 exons. Comparisons of the sequences and genomic organization of AtC401 and PnC401 revealed that each has two exons near the 3'-end, which encode a highly conserved domain consisting of 12 repeats of the PPR motif. Phylogenetic analysis of at least 450 Arabidopsis proteins containing PPR motifs revealed that AtC401 and related proteins form a distinct group. Moreover, the position of the intron between the two exons that encode the PPR domain has been conserved exactly in other C401-like genes. Using a reporter assay, we found a fragment (-174 to +73) of AtC401 that was sufficient to regulate circadian rhythmic expression. These results suggest that the conserved domain of AtC401 has a function similar to that of PnC401, and that the expression of C401 genes according to a circadian rhythm is important for protein function.