The optimum conditions for maximum oxidation of palmityl-carnitine by mitochondria isolated from the brown adipose tissue of 10-day-old rats was studied. The findings were as follows: 1. Reduction of the sucrose osmolar concentration to below 100 mM activates the rate of palmityl-carnitine oxidation, the maximum effect being achieved with 25 mM sucrose. These hypotonic conditions lead to enlargement of the matrix compartment, but not to swelling of the whole mitochondria. The maximum respiration rate in 25 mM sucrose can be measured only with fresh mitochondria isolated less than 1 hour previously, which must be preincubated 5 minutes in hypotonic sucrose before adding palmityl-carnitine. 2. When inducing the maximum palmityl-carnitine oxidation rate in 100 mM KC1 medium the preincubation time must be prolonged to at least 8 minutes. The length of time for which the mitochondria are stored in isotonic sucrose at 0 degrees C does not affect the respiration level in the presence of K+ ions. 3. The optimum palmityl-carnitine concentration is the same for oxidation measured in hypotonic sucrose and in KC1 medium and ranges from 15 to 50 muM. 4. If the above conditions are observed, the maximum palmityl-carnitine respiration values in hypotonic sucrose and medium with K+ ions are the same, whereas in isotonic sucrose respiration is inhibited. The same applies to the oxidation of endogenous fatty acids by the carnitine route and to alpha-ketoglutarate respiration, while the oxidation of alpha-glycerolphosphate is not affected by the osmotic conditions and its respiration is the same in both hypotonic and isotonic sucrose media.