BACKGROUND: Studies were conducted to elucidate the mechanism of glufosinate resistance in an Italian ryegrass population. RESULTS: Glufosinate rates required to reduce growth by 50% (GR(50)) were 0.15 and 0.18 kg AI ha(-1) for two susceptible populations C1 and C2 respectively, and 0.45 kg AI ha(-1) for the resistant population MG, resulting in a resistance index of 2.8. Ammonia accumulation after glufosinate treatment was on average 1.5 times less for the resistant population than for the susceptible populations. The glufosinate concentrations (µM) required to reduce the glutamine synthetase (GS) enzyme activity by 50% (I(50)) were 31 and 137 for C1 and C2 respectively, and 2432 for the resistant population MG. One amino acid substitution in the plastidic GS2 gene, aspartic acid for asparagine at position 171, was identified in the resistant population. CONCLUSIONS: This is the first report of glufosinate resistance in a weed species that involves an altered target site.