The development of acetylcholine receptor (AChR) and its clustering were studied quantitatively on mouse myotubes in nerve-muscle co-cultures. AChR was visualized by fluorescence-labeled antibodies (F-Ab) against crude AChR or fluorescence-labeled alpha-bungarotoxin (F-alpha-BuTX). The F-Ab stain was observed throughout the entire surface of the myotube at day 8 and appeared clustered at day 13. Both peak density and total amount of fluorescence in F-Ab stained myotubes were plotted against days in culture. Both fluorescence indices markedly increased from days 8 to 13 of culture were greater in extent in myotubes incubated in the presence of spinal cord explant, as compared with its absence. Similar results were observed in myotubes stained with F-alpha-BuTX. D-Tubocurarine (D-TC, 0.1 mg/ml) and native alpha-BuTX (1 microgram/ml) clearly inhibited both the total amount of fluorescence and the development of peak fluorescence density in the F-Ab stained myotubes. But the inhibition by D-TC appeared at the later day in culture than alpha-BuTX did. Low temperature (28 degrees C) and cholesterol (1 microgram/ml) treatment inhibited peak fluorescence density without affecting total amount of fluorescence. These results show that the development of ACh can be characterized both by clustering peak density (indicating the lateral mobility of AChR) and by total amount of fluorescence.