Two strategies have been used to increase the Tc-99(m) binding strength of human serum albumin (HSA) and thus enhance its blood retention. In a first approach, HSA was derivatized with a varying number of hydrazino nicotinyl (Hynic) side-chains using N-hydroxysuccinimidyl hydrazino nicotinate. Labelling of this albumin derivative with Tc-99(m) resulted in labelling yields of 90-95%. On the other hand, a Tc-99(m)-MAG3-HSA conjugate was prepared using the preformed chelate approach. In this way, non-specific binding of Tc-99(m) to HSA could be excluded. The in vitro stability of both Tc-99(m)-HSA derivatives was evaluated by cysteine challenge experiments, and revealed a much higher stability for Tc-99(m)-Hynic-HSA than for Tc-99(m)-MAG3-HSA. The biological behaviour of the preparations was evaluated in mice and a rabbit using I-125-HSA as an internal biological standard. The blood retention of Tc-99(m)-MAG3-HSA decreased more rapidly than that of I-125-HSA in both animal species, whereas Tc-99(m)-Hpnic-HSA seemed to provide a quasi-perfect Tc-99(m)-labelled,analogue for I-125-HSA and Tc-99(m)-red blood cells (Tc-99(m)-RBCs). In addition, the blood retention of Tc-99(m)-Hynic-HSA appeared to;be similar to that of Tc-99(m)-RBCs in a volunteer. These results dearly indicate the superiority of Tc-99(m)-Hynic-HSA over Tc-99(m)-MAG3-HSA as a possible blood pool agent.