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A phantom investigation was performed to validate a method of quantitative conjugate imaging designed to determine the activities in two separate aligned volumes at known depths within a uniformly attenuating medium. The method requires the acquisition of flood, transmission, and emission data from two opposing planar views. The flood data determine the camera sensitivities; the transmission data, the imaged object effective attenuation map; and the emission data, in conjunction with the object thickness and the depths of the two volumes, the activities in the volumes and the surrounding medium. Three experiments were conducted using technetium-99m (Tc-99m) in a 20.32-cm (8") diameter cylindrical phantom and a 58.75-cm × 43.0-cm rectangular flood tank. Two hollow spherical inserts having outer volumes of 16.2 ml were positioned symmetrically about the axis of the cylinder and with separation 10.16 cm (4"). Imaging was done using two heads of a Marconi IRIX triple-head system. A 15% energy window, symmetric about the Tc-99m photopeak, was used. Activity was injected into the phantom background and the spherical inserts such that the concentration ratios for sphere1:sphere2:background for the three experiments were 9:9:1, 12:6:1, and 15:3:1. Activity diluted at a single concentration in a 1-cc syringe was distributed by weight to ensure that activities placed in the phantom, spheres, and flood/transmission source were known to within better than 1%. Activities were low enough that camera dead-time effects were negligible. For each experiment, two flood data images were acquired simultaneously for 30 minutes; two transmission/emission data images, for 15 minutes each; and two emission data images, simultaneously for 15 minutes. MATLAB code was developed and used to estimate the effective attenuation coefficient of the medium, to compute the background activity concentration, and to compute the activity above background in each of the spheres. The method applied to the data acquired in the three experiments produced an overestimate of 2.5 to 12.2% for the larger above-background activity and an underestimate of 28.0% to an overestimate of 2.2% for the smaller above-background activity. In the three experiments, the sum of- the two activities was overestimated by 2.3 to 11.1%.