The important role of nucleotides in cellular metabolism requires that serious consideration be given to the question of the homogeneity or inhomogeneity of nucleotide pools in cells. The purpose of this review is to summarize the existing evidence for compartmentation of nucleotide pools, discuss the limitations of this evidence, and to discuss the implications of compartmentation for the interpretation of nucleotide concentration measurements. Evidence for nucleotide compartmentation comes from the following types of evidence: compartmentation of RNA precursors; compartmentation of deoxynucleoside triphosphates; mitochondrial compartmentation; the existence of tightly bound nucleotides; pools derived from alternative synthetic routes; compartmentation in cyclic nucleotide metabolism; channeling in the synthesis of pyrimidine nucleotides; and others. The types of evidence adduced for compartmentation will be considered critically and in detail, and alternative explanations considered, as well. Implications of the data and hypotheses on nucleotide compartmentation for the interpretation of nucleotide pool measurements in various types of experiments will be discussed.