The increased use of radiotelemetry for studying movement, resource selection, and population demographics in reptiles necessitates closer examination of the assumption that radiotransmitter attachment does not bias study results. We determined the effects of radiotransmitter attachment on fecal glucocorticoid metabolite levels of wild three-toed box turtles (Terrapene carolina triunguis) in captivity. During May 2002 we captured 11 adult three-toed box turtles in central Missouri. We housed turtles in individual pens in a semi-natural outdoor setting. We radiotagged 6 turtles, and the remaining 5 turtles served as controls. We captured and handled all turtles similarly during treatments. We collected feces daily prior to attachment (14 June–05 July 2002), while transmitters were attached (06 July–02 August 2002), and after transmitters were removed (03 August–24 August 2002). We conducted a standard assay validation and found that the assay accurately and precisely quantified fecal glucocorticoid metabolites of box turtles. We did not find a significant effect of radiotransmitter attachment on fecal glucocorticoid metabolite levels of three-toed box turtles (F_1,9 =0.404, P = 0.541). Fecal glucocorticoid metabolite levels of control and treatment turtles increased significantly during the study (F_2,166 =7.874, P = 0.001), but there was no treatment:period interaction (F₂, 166 = 0.856, P = 0.427). Additionally, we did not find a significant relationship between glucocorticoid metabolite levels and time in captivity (r² = 0.01, F_1,179 = 2.89, P = 0.091) or maximum daily temperature (r²<0.01, F_1,179 = 0.301, P = 0.584). Our results suggested that radiotransmitter attachment did not significantly increase fecal glucocorticoid metabolite levels in adult three-toed box turtles; however, we conducted our study in captivity and sample sizes were small. Thus, more research is needed to assess potential effects of radiotransmitters on turtles in the wild. We believe this study is the first to validate the use of fecal glucocorticoid metabolite measures for reptiles, which might prove useful in other research studies.