We evaluated factors affected the accuracy and precision of quantitating trace concentration of Ca with electron energy loss spectroscopy (EELS). These factors include internal reflection in the spectrometer, precision of correlation between standards and experimental spectra, and radiation damage-induced spectral changes. We present methods of correcting for these effects and improving the reliability of trace Ca quantitation. A two-step fitting procedure is described that improves the retrieval of small Ca signals from the large background common to biological specimens. After optimizing the experimental conditions and data processing procedures, our current system can detect about 2.2 mmol/kg Ca in a 730-Å thick specimen at a total dose of about 410 nA . sec at 95% confidence level by fitting the first difference spectra. Because of the 0.1% residual gain variation after gain normalization, the first difference spectrum fitting is still the preferred method for trace Ca quantitation. Our study also demonstrates the clear advantage of using a 200 keV system, instead of 120 keV or lower accelerating voltages, for EELS analysis of relatively thick biological cryosections.