Cells must abide by a number of constraints. The environmental constraints of cellular behavior and physicochemical limitations affect cellular processes. To regulate and adapt their functions, cells impose constraints on themselves. Enumerating, understanding, and applying these constraints leads to a constraint-based modeling formalism that has been helpful in converting conceptual models to computational models in biology. The continued success of the constraint-based approach depends upon identification and incorporation of new constraints to more accurately define cellular capabilities. This review considers constraints in terms of environmental, physicochemical, and self-imposed regulatory and evolutionary constraints with the purpose of refining current constraint-based models of cell phenotype. (C) 2003 Wiley Periodicals, Inc.