In a search for more selective anticancer drugs, we have designed nitrogen mustard and nitrosourea conjugates leading to a series of N-4-aryl-N'-2-chloroethylureas (CEUs). The iodinated derivative N-4-iodophenyl-N'-2-chloroethylurea (4-ICEU) has demonstrated significant antineoplastic and antiangiogenic potency in preclinical evaluations. In this study, 4-ICEU was radiolabelled with [(125)I]iodide in order to carry out a comparative study of its in vivo behavior profile. 4-[(125)I]-ICEU was synthesized by direct electrophilic radioiodination with 80% radiochemical yield and 97% radiopurity. Three different routes of administration (intraperitoneal (ip), intravenous (iv) and intratumoral (it)) were tested in mice bearing subcutaneously implanted CT-26 murine colon carcinoma. The results clearly established that 4-ICEU was more stable to biotransformation than previously studied CEUs congeners. It was readily bioavailable and reached the CT-26 colorectal tumor regardless of the route of administration. Additionally, the colon mucosa was an important target tissue where 4-ICEU accumulated and remained largely untransformed. In conclusion, these results justify further investigations for developing 4-ICEU as a new chemotherapeutic agent for colorectal cancer.