Abstract 1. The P 50 of whole untreated Carcinus mediterraneus haemolymph was found to be 9·5 ± 0·8 (S.E.M) mm HgO 2. 2. Dialysis against and dilution with Tris—HCl buffer, pH 7·6, resulted in a decreased O 2 affinity. 3. This decrease in affinity was shown not to be due to low Ca 2+ concentration or ionic strength. 4. Fractionation of whole haemolymph on Sephadex G-25 also resulted in a decrease in O 2 affinity. 5. O 2 affinity was found to be restored by the addition of haemolymph supernatant.