AbstractWe have characterized the highly detailed internal features of acetylcholine receptor (AChR) clusters on cultured rat myotubes by applying a novel image analysis technique—spatial fluorescence autocorrelation. AChR clusters were observed with total internal reflection fluorescence (TIRF) under high magnification after labelling the AChRs with tetramethylrhodamine α‐bungarotoxin, and recorded as CCD images. AChR clusters (on the tens of microns size scale) appear to be composed of microclusters (on the tenths of micron size scale), and the microclusters are sometimes organized into paracrystalline arrays. These structural features may reflect those of a protein meshwork that may underlie each cluster. To characterize these features, we have applied a spatial fluorescence autocorrelation technique which allows us to extract six parameters from each cluster image: average size of microclusters, contrast of microclusters, average spatial period of the paracrystalline arrays, contrast of periodicity, average fluorescence intensity, and average anisotropy of microclusters. These parameters were then followed as a function of myotube age after plating, and upon treatments with drugs which previously have been shown to gradually eliminate clusters. The drugs used were oligomycin (an energy metabolism inhibitor) and carbachol (a nicotinic AChR agonist). We show that myotube age only slightly affects the image parameters based on the spatial autocorrelation. Oligomycin induced significant decreases in the size of microclusters, contrast of microclusters, spatial period, contrast of periodicity, and anisotropy, whereas the carbachol study showed significant increases in the size of the microclusters and the anisotropy of the microclusters. These results suggest that these two drugs may act through different mechanisms in eliminating clus