Low-temperature phosphorescence (LTP) decay measurements have been carried out in wild-type Escherichia coli alkaline phosphatase (AP), metal-depleted AP (apoAP), and terbium-substituted AP (TbAP) at 77, 4.2, and 1.2 K. Over this temperature range, Tb emission monitored at 542.4 nm decays with two apparent lifetime components of 16-20 ms and 130-170 ms when TbAP is excited at 280 nm where Tb absorbs negligibly. These lifetimes are orders of magnitude greater than exhibited by TbCl3 at low temperature and by TbAP at room temperature; we attribute this Tb luminescence in TbAP largely to energy transfer from the triplet state of initially excited tryptophan. LTP and ODMR at 1.2 K suggest that Trp(109) and Trp(220) have similar environments in AP and in apoAP. Trp(109) is found to occupy two distinct sites in both AP and apo AP that exhibit resolved ODMR transitions. Comparison of the phosphorescence spectra of AP and TbAP shows clearly that the sensitized Tb emission observed in TbAP is due selectively to energy transfer (ET) from Trp(109) to Tb and not significantly from Trp(220). Evaluation of the results also suggests that ET takes place through space by Dexter's exchange mechanism, where the lowest triplet state of Trp acts as donor and the F-7(6) ground state of Tb acts as acceptor. The ratio of the two ET rate constants suggests that two sites of Trp(109) (possibly those resolved by ODMR) act as donors to either or both metal-binding sites, M2 or M3, rather than one Trp(109) site transferring energy to Tb at either site M2 or M3.