This study describes a quantitative method to measure the magnitude and distribution of gene expression following local delivery of an adenoviral vector containing the human sodium iodide symporter (hNIS) reporter gene into the canine prostate. Following systemic administration of Na(99m)TcO(4), autoradiographs of prostate sections depicting hNIS-dependent (99m)TcO(4)(-) uptake were digitized and stacked to produce a three-dimensional reconstruction of gene expression. Frequency histograms reflecting hNIS gene expression magnitude and volume were used to quantify hNIS function. The method demonstrated submillimeter resolution allowing for precise measurements of gene expression magnitude and volume in vivo. The method developed here could be applied to other reporter gene systems in which the readout can be digitized from thin tissue sections.