Intracellular potassium activity in guinea pig left atria was measured using potassium ion-selective microelectrodes and conventional microelectrodes. The effects of extracellular potassium concentration and acetylcholine on both intracellular potassium activity and the relationship between the resting membrane potential and the potassium equilibrium potential were investigated. Intracellular potassium activity was 102.1 mM in bathing media with a potassium concentration of 5 mM. Neither increasing extracellular potassium concentration to 10 mM nor exposure to acetylcholine (2 x 10(-6) to 10(-3) M) significantly altered intracellular potassium activity. In contrast, intracellular potassium activity decreased to 92.9 mM in 2.5 mM potassium concentration solutions. Resting membrane potential was 18.6, 9.6, and 7.3 mV positive to the potassium equilibrium potential in 2.5, 5, and 10 mM potassium, respectively. Acetylcholine caused a significant hyperpolarization at each extracellular potassium activity, confirming that resting membrane potential was positive to the potassium equilibrium potential. Even after exposure to 10(-3) M acetylcholine, the resting membrane potential apparently remained positive to the potassium equilibrium potential. If potassium accumulates in extracellular clefts during acetylcholine exposure, the calculated potassium equilibrium potentials are too negative, and the resting membrane potential might closely approximate the potassium equilibrium potential under these conditions. Fading of the acetylcholine-induced hyperpolarization and overshoot of the resting membrane potential on washout of acetylcholine were observed and are consistent with an accumulation of potassium during exposure to acetylcholine. In 5.0 mM potassium bathing solution, preparation-to-preparation variability of resting membrane potential can largely be explained by variability of intracellular potassium activity.(ABSTRACT TRUNCATED AT 250 WORDS)