Isometric contractions and cellular action potentials (A.P.) of isolated left atria of guinea pig hearts were recorded. The preparations were exposed to acetylcholine (ACh 1X10(-6) M), rapidly fixed at various functional states with 1% osmium tetroxide in 2% potassium antimonate and inspected with electron microscope. In the atria fixed at a constant stimulation rate of 90/min, calcium precipitates formed dense lines across the I bands of sarcomeres and were dispersed over A bands and mitochondria. They disappeared completely or almost completely from the atria perfused with calcium-free and containing EDTA or EGTA solutions. Precipitates were strikingly depleted in all locations in the atria treated with ACh at constant stimulation rate (which developed inotropic effect). ACh did not reduce relative density of precipitates in the resting atria. If stimulated, they developed only slightly diminished post-rest contraction. The relevance of these findings to the excitation-contraction coupling and to the mechanism of negative inotropic effect of ACh is discussed.