The molecular basis of spectral tuning of rhodopsin pigments in two squid species is examined. The absorbance spectra of rhodopsin extracts from Alloteuthis subulata (lambda max 499 nm) showed a 5 nm red shift compared with Loligo forbesi (lambda max 494 nm). The rhodopsin gene sequence of A. subulata opsin was determined from polymerase chain reaction (PCR) amplified fragments by using genomic DNA as template. The deduced amino acid sequence was compared with that obtained from the previously published cDNA sequence of Loligo forbesi. A total of 22 amino acid differences are present, although only seven can be considered to be non-homologous substitutions. Three of these changes occur in helical transmembrane regions but only one, the substitution of phenylalanine by serine at site 270 in Alloteuthis subulata, involves the replacement of an apolar with a hydroxyl-bearing residue at a site located near the centre of helix VI and on the inner face of the retinal-binding pocket. The equivalent site is also used for the spectral tuning of primate cone pigments, an example of convergent evolution. It is proposed that substitution at this site is responsible for the 5 nm red shift. The relation between this spectral shift and the maximum depth distribution of Alloteuthis subulata of about 200 m, compared with about 360 m for Loligo forbesi, is discussed. An unexpected finding was that the rhodopsin gene of both species appears to lack introns.