A novel orange fluorescent protein (OFP) was cloned from the tentacles of Cnidarian tube anemone Cerianthus sp. It consists of 222 amino acid residues with a calculated molecular mass of 25.1 kDa. A BLAST protein sequence homology search revealed that native OFP has 81% sequence identity to Cerianthus membranaceus green fluorescent protein (cmFP512), 38% identity to Entacmaea quadricolor red fluorescent protein (eqFP611), 37% identity to Discosoma red fluorescent protein (DsRed), 36% identity to Fungia concinna Kusabira-orange fluorescent protein (KO), and a mere 21% identity to green fluorescent protein (GFP). It is most likely that OFP also adopts the 11-strand beta-barrel structure of fluorescent proteins. Spectroscopic analysis indicated that it has a wide absorption spectrum peak at 548 nm with two shoulders at 487 and 513 nm. A bright orange fluorescence maximum at 573 nm was observed when OFP was excited at 515 nm or above. When OFP was excited well below 515 nm, a considerable amount of green emission maximum at 513 nm was also observed. It has a fluorescence quantum yield (Phi) of 0.64 at 25 degrees C. The molar absorption coefficients (epsilon) of folded OFP at 278 and 548 nm are 47,000 and 60,000 M(-1) x cm(-1), respectively. Its fluorescent brightness (epsilon Phi) at 25 degrees C is 38,400 M(-1) x cm(-1). Like other orange-red fluorescent proteins, OFP is also tetrameric. It was readily expressed as soluble protein in Escherichia coli at 37 degrees C, and no aggregate was observed in transfected HeLa cells under our experimental conditions. Fluorescent intensity of OFP is detectable over a pH range of 3 to 12.