The effect of spatial resolution on quantification by single photon emission computed tomography (SPECT) was studied using a rotating gamma camera and 99mTc. Using phantoms with hot and cold regions, experiments were performed to ascertain relationships between the source and the SPECT image, and to compare them with theoretic calculations. According to the results, the SPECT value represented the true radioactivity when the objects' sizes were 2.5 times larger than full width at half maximum (FWHM) for hot regions. For cold regions, there were errors of approximately 20% for the true value, even in the case of such large sizes. In addition, sizes at half maximum images corresponded to true object sizes when hot region sizes were larger than 1.4 x FWHM. The relationship between absolute radioactivity and total SPECT value was linear when the threshold level was zero. Knowing the effect of spatial resolution is a necessity in clinical SPECT studies.