Ammonium is the preferred nitrogen source and chemoattractant for the unicellular green alga Chlamydomonas reinhardtii. Here we describe that chemotaxis to methylammonium, a non-metabolizable analogue to ammonium, that occurs only in vegetative cells and pregametes but not in gametes. A new methylammonium-resistant mutant, hat1 that was generated by insertional mutagenesis, has been isolated and found to be affected at multiple loci. At the physiological level, hat1 showed altered Km and Vmax for high-affinity ammonium and methylammonium transport. Transcript levels of the ammonium transporter genes CrAmt1.(1-8) were similar to the wild type except for a lower expression level of CrAmt1.5, 6 and 8. Treatment with the potassium-channel inhibitor tetraethylammonium (TEA) blocked chemotaxis to ammonium/methylammonium and [14C]-methylammonium uptake. Our results suggest that the hat1 mutant could be affected in genes encoding regulatory elements of AMT1 transporter activities and that chemotaxis to ammonium/methylammonium is mediated by ammonium transporters sensitive to the potassium-channel inhibitor TEA.