As part of the long-term follow-up of amblyopic children who were enrolled in the Amblyopia Treatment Study, we tested visual acuity in both eyes of 142 patients by using 2 established visual acuity protocols, the Amblyopia Treatment Study HOTV (ATS HOTV) visual acuity protocol and the Electronic-Early Treatment of Diabetic Retinopathy Study (E-ETDRS) protocol, at one of the annual visits. Mean age at the time of testing was 9.0 years (range, 5.6-11.9 years). The ATS-HOTV protocol resulted in a slight mean overestimate of visual acuity relative to the E-ETDRS protocol (0.68 lines for amblyopic eyes; 0.25 lines for fellow eyes). The overestimation occurred primarily when the E-ETDRS visual acuity was poorer than 0.3 logMAR. ATS-HOTV acuity also underestimated interocular visual acuity differences by 0.42 lines. When one uses the ATS-HOTV protocol, it may be wise to exercise caution when interpreting "near-normal" visual acuity or interocular differences as "within normal" because the ATS-HOTV method may have a slight bias toward better performance of amblyopic eyes than E-ETDRS.