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Efforts to create dedicated nuclear medicine breast imaging scanners are maturing as they approach more routine clinical use. These systems promise to improve the detection of small lesions, especially those in breasts that are not definitively imaged with standard methods. Since these techniques lack sufficient specificity for accurate diagnoses of cancer, a method for guiding biopsies with these new devices must be developed. In this investigation, a technique that capitalizes upon the three-dimensional, multi-angle positron emission tomography (PET) breast images created by our new positron emission mammography/tomography imaging and biopsy device (PEM-PET) to guide breast biopsies was tested. Specifically, the accuracy and precision of the needle positioning system was measured for each of the three axes of motion. In addition, a biopsy of a simulated breast lesion was performed under realistic imaging conditions. The results from the positioning tests revealed that the system had very good accuracy and precision in all three axes. The maximum positioning error was plusmn 0.27 mm, sufficient to obtain samples from most lesions detected with PEM-PET. In addition, the simulated biopsy was performed successfully on a single attempt in obtaining a sample from a 5-mm-diameter target sphere. This successful test moves the project closer to the next phase of development, initiation of limited human testing.