The stoichiometric limit to the biomass yield (maximal assimilation of the carbon source) is determined by the amount of CO2 lost in anabolism and the amount of carbon source required for generation of NADPH. This stoichiometric limit may be reached when yeasts utilize formate as an additional energy source. Factors affecting the biomass yield on single substrates are discussed under the following headings: Energy requirement for biomass formation (YATP). YATP depends strongly on the nature of the carbon source. Cell composition. The macroscopic composition of the biomass, and in particular the protein content, has a considerable effect on the ATP requirement for biomass formation. Hence, determination of for instance the protein content of biomass is relevant in studies on bioenergetics. Transport of the carbon source. Active (i.e. energy-requiring) transport, which occurs for a number of sugars and polyols, may contribute significantly to the calculated theoretical ATP requirement for biomass formation. P/O-ratio. The efficiency of mitochondrial energy generation has a strong effect on the cell yield. The P/O-ratio is determined to a major extent by the number of proton-translocating sites in the mitochondrial respiratory chain. Maintenance and environmental factors. Factors such as osmotic stress, heavy metals, oxygen and carbon dioxide pressures, temperature and pH affect the yield of yeasts. Various mechanisms may be involved, often affecting the maintenance energy requirement. Metabolites such as ethanol and weak acids. Ethanol increases the permeability of the plasma membrane, whereas weak acids can act as proton conductors. Energy content of the growth substrate. It has often been attempted in the literature to predict the biomass yield by correlating the energy content of the carbon source (represented by the degree of reduction) to the biomass yield or the percentage assimilation of the carbon source. An analysis of biomass yields of Candida utilis on a large number of carbon sources indicates that the biomass yield is mainly determined by the biochemical pathways leading to biomass formation, rather than by the energy content of the substrate.