We have studied the effect of monensin on microtubule-associated translocation of atrial secretory granules in 5-7-day-old cultures of atrial myocytes from adult rats. Atrial granules and microtubules were localized by immunofluorescent microscopy of myocytes double-labeled with primary antibodies against atrial natriuretic polypeptide (ANP) and alpha-tubulin. In control myocytes, fluorescence due to atrial granules was predominantly localized to the perinuclear region containing the Golgi complex. After exposure for 30 minutes to monensin (0.5-5.0 microM), myocytes transiently contained conspicuous linear arrays of atrial granules associated with cytoplasmic microtubules. Thereafter, ANP fluorescence accumulated in subsarcolemmal foci at the cell periphery, while perinuclear ANP fluorescence faded. The monensin-induced redistribution of atrial granules was observable in both serum-containing and serum-free media and was unaffected by reducing external Ca2+ to low values, by inhibition of sarcoplasmic reticulum Ca2+ release with ryanodine, or by both. The redistribution was prevented by pretreatment with nocodazole, which fragmented microtubules and scattered Golgi complexes and the associated atrial granules throughout the cytoplasm. Radioimmunoassay showed that monensin seemingly decreased the rate of ANP secretion into the medium from 0.15 to 0.11 fmol/(hr.myocyte). These results suggest that monensin turns on microtubule-associated translocation of atrial granules from the perinuclear areas to the cell periphery by modifying the interaction between microtubules and atrial granules. Monensin also promotes movement of atrial granules along the microtubules but does not accelerate the release of ANP.