s-Triazine-degrading bacterial strains were isolated from long-term simazine-treated agricultural soils of central Chile. The total heterotrophic bacteria of these agricultural soils (7 x 10⁶ CFU/g of dry soil) were not affected after simazine application on field. By enrichment in minimal medium containing simazine as the sole nitrogen source, the bacterial strains P51, P52 and C53 were isolated and selected based on their simazine-degrading abilities. Resting cells of strains P51 and P52 degraded >80% of simazine within 48 hrs, whereas strain C53 was able to remove >60% of the herbicide. The atzA and atzD genes of the s-triazine catabolic pathways were detected in strains P51 and C53, while only atzD gene was observed in strain P52. To compare the bacterial 16S rRNA gene restriction pattern, ARDRA were performed using the restriction enzymes Msp1 and Hha1. ARDRA indicate that strain P52 is a different ribotype that C53 and P51 strains. For further characterization, the novel isolates were identified by 16S rRNA gene sequencing. Strains C53 and P51 belong to the genus Stenotrophomonas and the strain P52 belongs to the genus Arthrobacter. s-Triazine-degrading bacteria isolated from contaminated soils could be used as biocatalysts for bioremediation of these herbicides.