There is accumulating evidence that some biochemical pathways observable by magnetic resonance spectroscopy, e.g., citrate acid and phospholipid metabolism, are altered in human prostate cancer. Four well-established human prostate cancer cell lines were therefore studied with magnetic resonance spectroscopy to compare differences in metabolic content with tumor biological behavior. Herein we demonstrate that, although each cell line has its own metabolic profile, relative creatine and citrate levels can be used to discriminate the androgen-dependent LNCaP cell line from the androgen-independent DU-145, TSU, and PC-3 cell lines.