Inter-alpha-inhibitor, TSG-6, and hyaluronan have important functions in fertility and inflammation. Two subunits of inter-alpha-inhibitor, the heavy chains, form covalent bonds with TSG-6 or hyaluronan in vitro. TSG-6-heavy chain complexes serve as intermediates in the transfer of heavy chains from inter-alpha-inhibitor to hyaluronan. In vivo, in addition to these complexes, stable ternary complexes of hyaluronan with both TSG-6 and heavy chains have been demonstrated in the ovulatory cumulus oophorus. In our ongoing efforts to characterize the multiple interactions between hyaluronan, TSG-6 and inter-alpha-inhibitor, we recently characterized the formation of highly stable complexes of TSG-6 with hyaluronan that had been tethered to a solid surface. Here we show that these hyaluronan-TSG-6 complexes are functionally active and transfer heavy chain subunits from inter-alpha-inhibitor to either free or surface-bound hyaluronan. Transitional hyaluronan-TSG-6-heavy chain complexes do not accumulate in vitro. Our data show the capability for heavy chain transfer by both free TSG-6 and preformed hyaluronan-TSG-6 complexes, suggesting that both might contribute to hyaluronan modification in vivo. Transfer of heavy chains to surface-tethered hyaluronan by either free TSG-6 or surface-tethered hyaluronan-TSG-6 complexes did not affect the CD 44-mediated binding of BW 5147 cells in vitro. We show how TSG-6 and hyaluronan together can deplete inter-alpha-inhibitor and generate bikunin, as has been observed in sepsis, and discuss the role of TSG-6 in the generation of hyaluronan-heavy chain complexes associated with ovulation, arthritis, and sepsis.