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1,4-Dioxane (dioxane) is relatively recalcitrant to biodegradation, and its physicochemical properties preclude effective removal from contaminated groundwater by volatilization or adsorption. Through this microcosm study, we assessed the biodegradation potential of dioxane for three sites in California. Groundwater and sediment samples were collected at various locations at each site, including the presumed source zone, middle and leading edge of the plume. A total of 16 monitoring wells were sampled to prepare the microcosms. Biodegradation of dioxane was observed in 12 of 16 microcosms mimicking natural attenuation within 28 weeks. Rates varied from as high as 3,449 ± 459 µg/L/week in source-zone microcosms to a low of 0.3 ± 0.1 µg/L/week in microcosms with trace level of dioxane (<10 µg/L as initial concentration). The microcosms were spiked with (14)C-labeled dioxane to assess the fate of dioxane. Biological oxidizer-liquid scintillation analysis of bound residue infers that 14C-dioxane was assimilated into cell material only in microcosms exhibiting significant dioxane biodegradation. Mineralization was also observed per (14)CO2 recovery (up to 44% of the amount degraded in 28 weeks of incubation). Degradation and mineralization activity significantly decreased with increasing distance from the contaminant source area (p < 0.05), possibly due to less acclimation. Furthermore, both respiked and repeated microcosms prepared with source-zone samples from Site 1 confirmed relatively rapid dioxane degradation (i.e., 100 % removal by 20 weeks). These results show that indigenous microorganisms capable of degrading dioxane are present at these three sites, and suggest that monitored natural attenuation should be considered as a remedial response.