The objective was to study the enzymatic production of triiodothyronine (T₃) in the intestine of chickens during perinatal intestinal maturation in two lines of chickens selected for high (HW) or low (LW) body weight at eight weeks of age. Valid assay conditions (proportionality of enzyme activity with enzyme concentration and assay time) were established and the intestinal 5'-deiodinase (5'D) activity was characterized for comparison with other tissues. The characterization studies showed that intestinal 5'D is like the Type I 5'D in liver of birds and mammals previously studied. Specific activity of adult intestinal 5'D is significantly higher in the HW than in the LW line. In both lines intestinal 5'D increases significantly between embryos that have not pipped into the air cell (NP) and embryos that have pipped into the air cell (AC) and 5'D activity peaks in embryos that have pipped through the shell (TS). In contrast to the line differences in adults, LW embryos have much higher 5'D specific activity than HW embryos until 1d posthatch. Plasma thyroxine (T₄) and T₃ also increased between consecutive stages and peaked in embryos pipped through the shell, then decreased abruptly at 1d posthatch. Both plasma hormones were higher at each perinatal stage in the LW line than in the HW line and the LW line hatched earlier than HW. Intestinal alkaline phosphatase (a marker of differentiation) showed a significant increase in activity at each of the stages of development in both lines. Alkaline phosphatase activity was significantly higher in the LW line than the HW line at the NP, AC and TS stages but not at 1d posthatch. Previous work in other laboratories indicates that T₃ plays a role in triggering intestinal differentiation and maturation of intestinal function for posthatching life. The results of this study indicate that T₃ for this signal originates at least partially from 5'deiodination of T₄ within the intestinal tissues as well as from T₃ available in the plasma.