Previous studies of the serum binding of the photosensitizer hematoporphyrin (Hp) have given widely different results. The serum binding of Hp is therefore further illuminated by experiment and discussion. Ultracentrifugal separation of serum is improved and applied to study the binding of Hp to human serum and HSA. The observed distribution of Hp among the serum proteins is compared with the distribution expected from available association constants for Hp binding with individual proteins. The lipoprotein classes and the two major high density proteins (HDP), albumin and gamma-globulin, were separated in a NaCl-KBr gradient by single spin ultracentrifugation (SW 40; 30,000 rpm). HSA- and HDP-bound Hp were similarly distributed in the centrifuged gradient. Centrifugation of Hp-doped HSA separated the unbound Hp (75%) and the HSA-bound Hp (25%). The present association constant for the Hp-HSA complex (10(3)/M) was much lower than earlier published ones (10(6)/M) found by other techniques. The association of Hp with HDP in serum was much stronger than the association of Hp with the isolated HSA (electrophoretic grade). The estimated ratio of HSA-bound to LDL-bound HP in serum was at least 25 times larger than the experimental value. The percentage of LDL-bound Hp decreased with increasing Hp concentration. The serum binding of Hp is the same as that found previously using another rotor and another salt gradient (70.1 Ti, 70,000 rpm, NaCl-CsCl). LDL has high-affinity-low-capacity binding sites for Hp. HSA is the major HDP protein that binds Hp in human serum. The strength of the HSA-Hp complex may depend on the batch of HSA used and upon the absence/presence of other proteins. Proteins may interact in serum in manners that affect the binding of certain drugs. Neither the type of gradient salt nor the field of gravity affected the serum binding of Hp.