It has previously been reported that endophytic diazotrophic bacteria contribute significantly to the nitrogen budgets of some graminaceous species. In this study the contribution of biological nitrogen fixation to the N-budget of a South African sugarcane cultivar was evaluated using 15 N natural abundance, acetylene reduction and 15 N incorporation. Plants were also screened for the presence of endophytic diazotrophic bacteria using acetylene reduction and nifH-gene targeted PCR with the pure bacterial strains. 15 N natural abundance studies on field-grown sugarcane indicated that the plants did not rely extensively on biological nitrogen fixation. Furthermore, no evidence was found for significant N 2 -fixation or nitrogenase activity in field-grown or glasshouse-grown plants using 15 N incorporation measurements and acetylene reduction assays. Seven endophytic bacterial strains were isolated from glasshouse-grown and field-grown plants and cultured on N-free medium. The diazotrophic character of these seven strains could not be confirmed using acetylene reduction and PCR screening for nifH. Thus, although biological nitrogen fixation may occur in South African sugarcane varieties, the contribution of this N-source in the tested cultivar was not significant.Abbreviations: n 15 N -nitrogen isotope discrimination; d 15 N -nitrogen isotope composition.