Although laboratory experiments allow greater control of environmental conditions than field studies, they have several drawbacks. To analyze physiological responses to forcing environmental variables, experimental conditions should mimic natural conditions as closely as possible. For filter-feeding organisms in particular, diet quality and quantity is one of the environmental parameters that can differ markedly between experimental and field conditions. In the hatchery, Pacific oysters, Crassostrea gigas, commonly show good physiological performance and growth on a mixed algal diet of Tisochrysis lutea, formerly Isochrysis aff. galbana clone Tahiti (T-Iso), and Chaetoceros calcitrans, presumably as it provides a good supply of essential polyunsaturated fatty acids (PUFA) as 20:4n-6, 20:5n-3 and 22:6n-3. The present study tests whether the fluctuating biotic and abiotic conditions in the field modify the structure and function of oyster mitochondria. One group of oysters was maintained in the intertidal zone, and the other group was fed the mixed diet in a nearby experimental hatchery under salinity and temperature conditions equivalent to those in the field. After 4 weeks of conditioning, the functional capacities and membrane lipid composition of gill mitochondria were measured. For essential polyunsaturated fatty acids, only the proportion of 20:5n-3 differed between field and laboratory oysters, and confirmed the capacity of the mixed diet T-Iso + C. gracilis, to provide the essential PUFA. Nevertheless, proportions of other FA (e.g., 22:5n-6 and non-methylene-interrupted FA) differed markedly between laboratory and field-conditioned oysters. Mitochondrial oxygen uptake, cytochrome c oxidase activity, content of cardiolipin and concentration of cytochrome b were significantly lower in laboratory-conditioned than in field-conditioned oysters. These results indicate that laboratory conditioning, although allowing similar growth and gonad maturation, only partially mimics conditions that allow C. gigas to maintain mitochondrial function. Although our experimental design cannot ascertain what difference between experimental laboratory and field conditions led to changes in membrane composition and mitochondrial function, differences in nutritional quality (other than known essential PUFA) and abiotic factors (e.g., oxygen availability, emersion or daily temperature fluctuations) had a major impact on mitochondrial properties in oysters.