Cholecalciferol (D3) deficiency in animals and humans is associated with skeletal deformities and retarded growth. Hence, the evaluation of D3 sources is useful to ensure adequate biological activity. For this experiment, two sources of D3 were used to compare both biological activities using a chick bioassay. Newly hatched broiler chickens were caged in 2 battery brooders for a 17-day trial period. Experimental treatments were created from a common basal D3-deficient corn-soy broiler starter diet. The basal diet was supplemented with 0 (NC), 62.5, 125, 250, 500, or 1,000 IU D3/kg of feed. All birds were fed the basal diet devoid of D3 (NC) over the first 9 d to deplete the maternal D3, followed by a 12-hour fasting period. On d 10, the test diets were offered ad libitum for 7 days. Feed consumption, body weight (BW), and mortality were recorded to evaluate weight gain (WG) and feed efficiency (FE). Additionally, bone mineral content (BMC), bone mineral density (BMD), percent fat-free dried tibia ash (TBA), and tibia breaking strength (TBS) were used to evaluate bone mineralization. Relative bioavailability (RBV) of D3 was determined by the slope-ratio method using the powdered D3 as the reference standard source. Estimated RBV of the D3 beadlets source was 0.74, 0.75, 0.79, 0.80, 0.65, and 0.63 when BW, WG, TBA, TBS, BMC, and BMD were used as response criteria, respectively. In conclusion, this protocol was able to detect differences between highly concentrated D3 sources and may be employed to evaluate D3 biological activity. [ABSTRACT FROM AUTHOR]