Hexakis (2-methoxy isobutylisonitrile) technetium-99m [(99mTc]MIBI), a new tracer of myocardial blood flow, was compared with 201TI CI in cultures of myocardial cells of newborn rats. The kinetics of uptake and release of both tracers were assessed in basal conditions and in the presence of 5 mM cyanide, an inhibitor of the respiratory chain, 0.1 mM iodoacetate, an inhibitor of glycolysis, 10 microM ouabain, an inhibitor of the Na-K ATPase, or with various pH values. The amplitude and frequency of contractions of the cells were also monitored in the same conditions. Results show that the washin and washout kinetics of [99mTc]MIBI are slower than 201TI(T1/2) of the washout curves were, respectively, of 28 min and 6 min). The kinetics of release of both tracers were not influenced by any of the inhibitors. There was a strong effect of the pH on the 201TI uptake only. Moreover 201TI uptake was decreased by 34% in the presence of cyanide plus iodoacetate. Otherwise the uptakes of 201TI and [99mTc]MIBI were not decreased by any of the drugs. The cellular contractility was significantly diminished by cyanide and it was abolished by cyanide plus iodoacetate. It is concluded that (a) impaired contractility can be associated with normal 201TI and [99mTc]MIBI kinetics in myocardial cells in culture, (b) that 201TI uptake may depend on the level of ATP devoted to the maintenance of membrane integrity, (c) that [99mTc]MIBI shows slower kinetics but is less sensitive to metabolic inhibitors than 201TI.