An increase of cellular phosphocholine (PC) and total choline (tCho)-containing compounds as well as alterations in lipids have been consistently observed in cancer cells and tissue. These metabolic changes are closely related to malignant transformation, invasion, and metastasis. The study of cancer cells in culture plays an important role in understanding mechanisms leading to altered choline (Cho) and lipid metabolism in cancer, as it provides a carefully controlled environment. However, a solid tumor is a complex system with a unique tumor microenvironment frequently containing hypoxic and acidic regions and areas of nutrient deprivation and necrosis. Cancer cell-stromal cell interactions and the extracellular matrix may also alter Cho and lipid metabolism. Human tumor xenograft models in mice are useful to mimic the growth of human cancers and provide insights into the influence of conditions on metabolism. Here, we have compared metabolites, obtained with high resolution H MRS of extracts from human breast and prostate cancer cells in a 2-dimensional (2D) monolayer culture and from solid tumor xenografts derived from these cells, as well as the protein expression of enzymes that regulate Cho and lipid metabolism. Our data demonstrate significant differences in Cho and lipid metabolism and protein expression patterns between human breast and prostate cancer cells in culture and in tumors derived from these cells. These data highlight the influence of the tumor microenvironment on Cho and lipid metabolism.