A recently isolated strain of Beggiatoa, MS-81-6 (cf. alba), was tested for chemoautotrophic growth in semi-solid (0.2% agar) sulfide/oxygen gradient cultures. The organism grew in a horizontal layer, the distance from the air/medium interface depending on sulfide concentrations and changing with time. Optimal growth as a gradient organism was based on a preference for reduced oxygen concentrations and a limited sulfide tolerance in combination with gliding motility. In gradient cultures chemoautotrophic growth was demonstrated by the following criteria: (1) biomass yield (protein) increased with increasing sulfide concentration, and estimated molar growth yields agreed with those for other sulfide-grown chemoautotrophs; (2) approximately 90% of total cell carbon and protein carbon were fixed from carbon dioxide; (3) the CO2-fixing enzyme, ribulosebisphosphate carboxylase, was present in cell-free extracts at a level typical of chemoautotrophs; (4) acidification of the medium, apparently linked to utilization of internal So granules, accompanied the later phase of growth. The ability to grow on acetate in the absence of a source of reduced sulfur renders the organism facultatively chemoautotrophic.