Pegylated zinc protoporphyrin (PEG–ZnPP) is a water-soluble inhibitor of heme oxygenase-1. In this study, we prepared two types of PEG–ZnPP conjugates with different chemical bonds between PEG and ZnPP, i.e., ester bonds and ether bonds, where both conjugates also contain amide bonds. Cleavability of these bonds in vitro and in vivo, especially cancer tissue, and upon intracellular uptake, was investigated in parallel with biological activities of the conjugates. Each conjugate showed different cleavability by plasma esterases and tumor proteases, as revealed by HPLC analyses. PEG–ZnPP with ester bond (esPEG–ZnPP) was more sensitive than PEG–ZnPP with ether bond (etPEG–ZnPP) for cleavage of PEG chains. etPEG–ZnPP showed no cleavage of PEG chains and had lower intracellular uptake and antitumor activity than did esPEG–ZnPP. The degradation of esPEG–ZnPP appeared to be facilitated by both serine and cysteine proteases in tumor tissues, whereas it was significantly slower in normal organs except the liver. Depegylated products such as free ZnPP had higher intracellular uptake than did intact PEG–ZnPP. We also studied hydrolytic cleavage by blood plasma of different animal species; mouse plasma showed the fastest cleavage whereas human plasma showed the slowest. These results suggest that ester-linked conjugates manifest more efficient cleavage of PEG, and greater yield of the active principle from the conjugates in tumor tissues than in normal tissues. More efficient intracellular uptake and thus an improved therapeutic effect with ester-linked conjugates are thus anticipated with fain stability, particularly in human blood.