The purpose of this study was to investigate the effectiveness and safety of percutaneous pancreatic transplant biopsy guided by ultrasound alone or with a combination of computerized tomography (CT) for pancreas localization and ultrasound for needle placement. We also compare our finding on the use of 18-gauge and 20-gauge needles for percutaneous pancreatic transplant biopsy. In 42 attempted biopsies performed on 21 patients, two different imaging modalities were used. Twenty-seven attempted biopsies were performed under the guidance of ultrasound alone, and 15 used a combination of ultrasound and CT. Of the 27 ultrasound-guided biopsies. 24 produced at least one sample adequate for histopathological analysis for an 89% biopsy success rate. Of the 15 biopsies guided by combined ultrasound and CT, 11 produced adequate samples for a 73% success rate. For all biopsies, an 83% success rate was found. In assessing the use of 18-gauge versus 20-gauge needles, 86 out of 110 tissue cores were adequate for histopathological analysis for a 78% yield. In 27 biopsy attempts using the 18-gauge needle, 75 tissue cores were obtained, for an average of 2.8 cores per biopsy. Fifty-seven pancreas samples collected using the 18-gauge needle were adequate for pathological evaluation for a 76% yield. With 15 biopsy attempts using the 20-gauge needle, 35 tissue cores were collected, for an average of 2.3 cores per biopsy. Twenty-nine pancreas specimens obtained from using the 20-gauge needle were adequate for analysis for an 83% yield. No major complications occurred. Only one incidence of minor complication was reported for a 2% complication rate. The only complication was local, mild bleeding at the biopsy site in one case. Air within the transplant pancreas as revealed by post-biopsy scans and streaky density appearing adjacent to the biopsy site occurred in a total of four cases and were not included. No complications were reported that required any invasive intervention. We conclude that percutaneous biopsy guided by ultrasound is a safe, simple, and effective method to detect pancreatic transplant rejection. Our results for biopsies compare favorably with other reported techniques in terms of effectiveness, complication rates, and ease of use. With its high success rate and low complications, ultrasound-guided percutaneous biopsy is an excellent method to sample pancreatic transplant.