The stability of the sheep haemoglobin molecule (type B) has been investigated at neutral pH over a range of ionic strengths by osmotic pressure (
π
) measurements in phosphate buffer and unbuffered NaCl solutions at 5 and 22 °C. At low ionic strength (
I
< 0.10), no significant dissociation of the molecule could be detected and the molecular weight obtained (
M
= 64 100) was in good agreement with that obtained from amino acid sequence studies (64 500). At higher ionic strength (
I
> 0.10), reversible dissociation was detectable as a deviation from a straight line plot of
π/c
against
c
, and, using a graphical method as well as a computer program, the most probable values of the dissociation constant (
K
d
) (describing the dissociation,
α
2
β
2
⇌ 2
αβ
) and interaction constant (
B'
) were obtained. The dissociation constants increased significantly with increasing ionic strength up to
I
= 2, but thereafter remained approximately constant. A plot of the standard free energy of dissociation,∆
G
0
d
, against ionic strength was curved but extrapolated to a value of approximately 32 kJ /mol in good agreement with that found for human haemoglobin in the presence of a variety of dissociating reagents. At the higher ionic strengths (
I
= 2) and particularly at 22 °C, there was some evidence that dissociation proceeded further than
αβ
but further work is required to substantiate this finding.