A near-infrared (NIR) imaging system is evaluated as a diagnostic clinical tool to image total hemoglobin concentration and oxygen saturation within tissue. Calibration of this type of system requires measurement of the response at each detector and source location from a homogeneous tissue-simulating phantom. The effect of using calibration phantoms of varying composition, size, and optical properties is examined to determine how it affects the overall image accuracy. All of the calibration phantoms investigated result in accurate reconstruction of absorbing heterogeneities due to increased blood concentration with less than 4% standard deviation. Images from a patient with a biopsy-confirmed ductal carcinoma are also evaluated and found to be insensitive to the choice of calibration object, with only 1% variation between images generated with different calibration objects. The tumor total hemoglobin contrast is approximately 240% higher than the average total hemoglobin concentration in contralateral breast. Soft calibration phantoms, which mimic the elastic properties of human breast tissue, are also considered and found to diminish positioning errors in the fibers relative to the actual breast exam, thereby reducing the artifacts in the periphery of the reconstructed image. © 2003 Society of Photo-Optical Instrumentation Engineers.