The physiological basis of glufosinate resistance for two resistant (R) rice mutants, lines ‘R11-2’ and ‘R11-3’, was studied. Seven days after the application of 0.54 mM glufosinate, two susceptible (S) lines, i.e., variety (var.) ‘FSK’ and its inbred line ‘FSK-3’, and a reference var. Tainung 67 (TNG 67) suffered severe injury, whereas the two R lines exhibited resistance. Dose–response analysis and survival rate 14 d after treatment with 1.5 mM glufosinate also supported this observation. A 14C-glufosinate experiment showed that more labeled herbicide was absorbed by leaves of R11-2 than S lines 48 h after treatment (HAT), but the partitioning of absorbed glufosinate to each part of the shoot did not differ between R and S lines. Although a higher degradation of glufosinate in R line R11-2 was found as compared with the two S lines, i.e., 46% vs. 38 to 40%, the actual concentration of glufosinate in R line was still higher than that in S lines. Foliar application of glufosinate resulted in less inhibition of in vivo activity of glutamine synthetase (GS; EC 6.3.1.2) as well as a lower accumulation of ammonium 24 HAT in R line than in S lines. Further kinetic study of GS showed that cytosolic GS in line R11-2, with a higher enzyme-inhibition constant (Ki) value to glufosinate, was less sensitive to the toxic action of this herbicide. Therefore, a higher metabolism of, and more important, a lower susceptibility of, the target protein GS to this herbicide are suggested to contribute significantly to glufosinate resistance in these rice lines.