We report the infrared spectrum of the 4-aminobenzimidazole-1-methylthymine (4ABI:1MT) heterodimer, detected by femtosecond multiphoton ionization. Based on calculations of both the harmonic and the anharmonic frequencies, the observed vibrational spectrum is assigned to a structure that mimics the Hoogsteen base pairing of adenine and thymine. A notable observation made in the course of this study is that there is a significant imbalance in the observed strengths of the H-bonds. While the N center dot center dot center dot H-N bond reveals a large red shift of >700 cm(-1) for the NH stretch frequency, the N-H center dot center dot center dot O bond is characterized by only a 50 cm(-1) shift. The importance of this observation in the formation of Hoogsteen duplexes by thymine-based oligonucleotides is discussed.