Solvent extracts of three different prokaryotic algae from three species of didemnid ascidians contained pigments identified, on the basis of their fluorescence excitation (E) and fluorescence emission (F) spectral maxima (measured in nm) at 77 K, as chlorophyll a (E 449, F 678) and chlorophyll b (E 478, F 658). The release of algae on cutting or freezing Diplosoma virens was accompanied by a strong unidentified acid that converted these pigments to pheophytins. This unexpected finding provided further confirmation of the identity of the chlorophylls on the basis of the fluorescence spectra at 77 K of pheophytin a (E 415, F 669) and pheophytin b (E 439, F 655). There was no evidence for the presence of the fluorescent bilin pigments found in other prokaryotic blue-green algae. Chlorophyll a/b ratios ranged from 2.6 to 12.0 in algae from different ascidians. The photosynthetic membranes were not organized into appressed thylakoids or grana in the algae from any of the three species of ascidians. The relationship between these observations and those in higher eukaryotic organisms is discussed.Several species of colonial ascidians in the family Didemnidae (Phylum Chordata, Subphylum Urochordata) from the Great Barrier Reef of northeastern Australia harbor bright green, unicellular algae in their cloacal cavities. Earlier studies of these algae showed that they are prokaryotic, with an ultrastructure resembling that of blue-green algae, but that they apparently lack the phycobilin pigments and the phycobilosomes characteristic of the blue-green algae (1). Similar prokaryotic algae have been reported growing on the surface of an unidentified didemnid ascidian from Baja California, and the presence of chlorophylls a and b in these algae was provisionally established on the basis of chromatography and room temperature absorbance spectra (2,3 (Hartmeyer), Trididemnum Cyclops (Michaelsen), and juvenile colonies of Didemnum ternatanum (Gottschaldt) were collected at low tide from coral rocks immediately behind the reef front. Larger, older colonies of D. ternatanum were found attached to branches of staghorn coral in deeper water along the front edge of the reef. The specimens collected in July were identified at the Heron Island Research Station laboratory by Patricia Kott Mather. Samples were embedded as previously described (4) for later electron microscopic study.Differences were noted in the ease with which the algae are removed from their ascidian hosts. Those from Diplosona mrens were released readily by applying pressure to the ascidian colony after it had been cut in two. The algae from this ascidian were either expressed into cold, filtered sea water, or into cold 0.1 M Tris.HCl buffer at pH 8.3, and then sedimented by centrifugation. Algae were much more difficult to extract from the other two ascidians, and consequently were isolated in relatively small quantities from these species. In Didemnum ternatanum their release is accompanied by the breakage of many of the algal cells and by embedment of the ...