The bioactive molecule sphingosine-1-phosphate (S1P) binds with high affinity to five recognized receptors (S1P_1-5) to affect various tissues, including cellular responses of cardiac fibroblasts (CFb) and myocytes. CFb are essential components of myocardium, and study of their cell signaling and physiology is required for a number of emerging disciplines. Meaningful studies on CFb, however, necessitate methods for selective, reproducible cell isolations. Macrophages reside within normal cardiac tissues and often co-isolate with CFb. A protocol was therefore developed that significantly reduces macrophage levels and utilizes more CFb-specific markers (discoidin domain receptor 2), instead of, or in addition to, more commonly used cytoskeletal markers. Our results demonstrate that primary isolated, purified CFb express predominantly S1P_1-3; however, the relative levels of these receptor subtypes are modulated with time and by culture conditions. In co-culture experiments, macrophages altered CFb S1P receptor levels relative to controls. Further investigations using known macrophage-secreted factors showed that S1P and H₂O₂ had minimal effects on CFb S1P_1-3 expression, whereas TFG1, TNF, and PDGF-BB significantly altered all S1P receptor subtypes. Lowering FBS concentrations from 10% to 0.1% increased S1P₂, whereas supplementation with either PDGF-BB or Rho-associated protein kinase inhibitor Y-27632 significantly elevated S1P₃ levels. S1P₂ and S1P₃ receptor levels are known to regulate cell migration. Using cells isolated from either normal or S1P₃-null mice, we demonstrate that S1P₃ is important and necessary for CFb migration. These results highlight the importance of demonstrating CFb culture purity in functional studies of S1P and present conditions that modulate S1P receptor expression in CFb.