Biphalin (Tyr-D-Ala-Gly-Phe-NH)2 is a unique opioid peptide analog that contains two active enkephalin pharmacophores and is more potent than morphine and etorphine in eliciting analgesia after intrathecal administration. After systemic administration, only a small amount was detected in the brain, but analgesia was observed. Because halogenation of enkephalin analogs has been shown to increase the brain uptake after systemic administration, our research group synthesized both p-[Cl-Phe4,4']biphalin and p-[F-Phe4,4']biphalin. The aim of the present study was to characterize and compare the blood-to-central nervous system (CNS) pharmacokinetics and biological stability of biphalin and related halogenated analogs. The initial screening used an in vitro blood-brain barrier model and identified p-[Cl-Phe4,4')biphalin as the enkephalin analog with the best potential for greater CNS entry. The CNS uptake and stability of biphalin and p-[Cl-Phe4,4']biphalin was examined further using an in situ brain perfusion technique coupled to high-performance liquid chromatography analysis. Both biphalin and its chlorohalogenated analog, were found to significantly enter the CNS through both the blood-brain and blood-cerebrospinal fluid barriers. Chlorohalogenation of biphalin was shown to both improve CNS entry, most likely through an enhancement in lipophilicity, and increase biological stability. This study suggests that incorporation of chlorohalogens at the p-Phe4,4' position is a promising structural modification in the development of biphalin as a successful opioid drug for the clinic.