The radioactivity distribution of 75Se-labeled D-selenomethionine (D-SeMet-75Se) was investigated in tumor-bearing mice in order to develop a new radioactive diagnostic agent. D-SeMet-75Se was enzymatically prepared from commercially available L-selenomethionine-75Se (L-SeMet-75Se) by using amino acid reacemase and immobilized L-amino acid oxidase. No difference between D- and L-SeMet-75Se was found in the excretion rate into urine and feces in normal mice within 48 h after administration. The in vivo uptake of D-SeMet-75Se in both Ehrlich solid tumor and sarcoma- 180 solid tumor was several times higher than that of L-SeMet-75Se but the uptake of D- and L-SeMet- 75Se were similar in the pancreas. These results indicated that D-SeMet-75Se might be useful as a tumor-imaging agent. In vitro experiments on Ehrlich ascites tumor cells showed that D-SeMet-75Se was incorporated into the tumor cells by a Na+-dependent active transport system similar to L-SeMet-75Se and that a transport mechanism specific for D-SeMet-75Se might be present in tumor cells in addition to a transport system common to both D- and L-forms.