Concentrations of DIBOA [2,4-dihydroxy-1,4-(2H)-benzoxazine-3-one] and BOA [2-(3H)-benzoxazolinone], described previously as major allelochemicals in Secale cereale (rye), were determined in eight field-grown cultivars, harvested at booting, using high-performance liquid chromatography (HPLC). Allelochemicals were also quantified in greenhouse-grown cultivar ‘Bates’ harvested 30, 45, 60, and 75 days after planting (DAP). The total production of DIBOA and BOA from field-grown S. cereale ranged from 137 to 1,469 μg g−1 dry tissue among the eight cultivars. ‘Bonel’ cultivar had the highest hydroxamic acid (HA) content and ‘Pastar’ the lowest. Bonel also showed the highest activity on Eleusine indica (goosegrass) and Pastar the least, in culture plate bioassays using aqueous extracts. HA content in shoot tissue varied with S. cereale maturity. The greatest level of HA in greenhouse-grown Bates was obtained 60 DAP compared to 30 DAP.