Folic acid (folate; vitamin Bc) is well recognized as essential for the proper metabolism of the essential amino acid methionine as well as for the synthesis of adenine and thymine. A folate deficiency has been implicated in a wide variety of disorders from Alzheimer's disease to depression and neural tube defects. In the cellular slime molds, including Dictyostelium, vegetative growthphase cells are known to chemotactically move toward folate that is secreted by bacterial food sources such as Escherichia coli. Intracellular folate signal transductlon, including G proteins, Ca² channels, and the PIP3 pathway, has been reported in D. discoideum. To our surprise, the genuine chemoattractant(s) of free-living protozoan amoebae have remained to be determined, possibly because of lack of a pertinent method for assaying chemotaxis. We recently isolated a primitive free-living amoeba from the soil of Costa Rica and identified it as a new species of the genus Vahlkampfia belonging to Subclass Gymnamoebia, which includes Entamoeba and Acanthamoeba. The amoebae can grow and multiply quite rapidly, engulfing nearby bacteria such as E. coli. Importantly, we have demonstrated here using a quite simple but finely designed chemotaxis assay that the Vahlkampfia amoebae exhibit chemotaxis toward higher folate concentrations. Riboflavin and cyanocobalamin were also found to serve as positive chemoattractants. Among these chemoattractants, folate is of particular importance because its function seems to be evolutionarily conserved as a potent chemoattractant of amoeboid cells in a wide range of organisms as well as in the Protista and cellular slime molds.