Ammonium is an important source of nitrogen for many microorganisms, including yeast, and its availability also has substantial effects on the nitrogen metabolism and development of yeast cells. Three ammonium transporter genes of the fission yeast Schizosaccharomyces pombe, named amt1, amt2, and amt3, were identified on the basis of amino acid sequence similarity to members of the ammonium transporter/methylammonium permease (Amt/Mep) family. A series of strains were constructed that carry all combinations of amt deletion (amt delta) mutations, and tested for growth on low ammonium and resistance to the toxic ammonium analog methylammonium. The amt1 delta and amt2 delta single mutants had different growth defects, and the amt1 delta amt2 delta double mutant displayed a much more severe growth defect on < or = 5 mM ammonium. All single mutants exhibited methylammonium resistance but to different extents: amt2 delta was the most resistant and amt3 delta was the least. These results suggest that the amt genes encode functional transporters with distinct uptake properties. In response to ammonium limitation, the wild-type strain isogenic to the amt delta mutants underwent filamentous growth underneath the surface of solid medium. No such filamentous invasive growth, however, was observed for the amt1 delta mutant, indicating that Amt1 transporter is required for ammonium limitation-induced filamentous invasive growth.