In the bacterial ISC system for iron-sulfur cluster assembly, IscU acts as a primary scaffold protein, and the molecular co-chaperones HscA and HscB specifically interact with IscU to facilitate ATP-driven cluster transfer. In this work, cluster transfer from Azotobacter vinelandii [Fe(2)S(2)](2+) cluster-bound IscU to apo-Grx5, a general purpose monothiol glutaredoxin in A. vinelandii, was monitored by circular dichroism spectroscopy, in the absence and in the presence of HscA/HscB/Mg-ATP. The results indicate a 700-fold enhancement in the rate of [Fe(2)S(2)](2+) cluster transfer in the presence of the co-chaperones and Mg-ATP, yielding a second-order rate constant of 20 000 M(-1) min(-1) at 23 °C. Thus, HscA and HscB are required for efficient ATP-dependent [Fe(2)S(2)](2+) cluster transfer from IscU to Grx5. The results support a role for monothiol Grx's in storing and transporting [Fe(2)S(2)](2+) clusters assembled on IscU and illustrate the limitations of interpreting in vitro cluster transfer studies involving [Fe(2)S(2)]-IscU in the absence of the dedicated HscA/HscB co-chaperone system.