The effect of ion space-charge on mass accuracy in Fourier transform ion cyclotron resonance mass spectrometry is examined. Matrix-assisted laser desorption/ionization is used to form a population of high-molecular-weight polymer ions with a wide mass distribution. The density of the ions in the analyzer cell is varied using ion remeasurement and suspended trapping techniques to allow the effect of ion space charge to be examined independently of other experimental influences. Observed cyclotron frequency exhibits a linear correlation with ion population. Mass errors of 100 ppm or more in externally calibrated mass spectra result when ion number is not taken into account. By matching the total ion intensities of calibrant and analyte mass spectra, the protonated ion of insulin B-chain, 3494.6513 Da, is measured with an accuracy of 0.07 ppm (average of 10 measurements, σ = 2.3 ppm, average absolute error 1.6 ppm) using a polymer sample as an external calibrant. Alternatively, the correction for space charge can be made by using a calibration equation that accounts for the total ion intensity of the mass spectrum. A calibration procedure is proposed and is tested with the measurement of the mass of insulin B-chain. A mass accuracy of 2.0 ppm (average of 20 measurements, σ = 4.2 ppm, average absolute error 3.5 ppm) is achieved. Space-charge-induced mass errors are more significant for samples with many components, such as a polymer, than for single-component samples such as purified peptides or proteins.