The quantity and spatial distribution of calcium bound to myofilaments is of considerable interest to physiologists studying the regulation of contraction in striated muscle, but few techniques allow these distributions to be directly measured. Methods are described here by which such data can be derived from electron probe X-ray microanalysis (EPXMA) images. Combination of data from multiple half sarcomeres improves the signal-to-noise level and allows assessment of variation between half sarcomeres. Profiles of calcium counts, calcium counts normalized to local bremstrahlung counts, and calcium counts normalized to the average bremstrahlung counts for the entire half sarcomere provide differing but complementary information. Such data help to identify and characterize spatial variations in Ca binding to regulatory proteins in muscle.