Sequential immobilization of single-stranded complementary DNA and peptide nucleic acid (PNA) molecules at the air-water interface with cationic Langmuir monolayers is demonstrated. The complexation of the single-stranded DNA/PNA molecules with octadecylamine (ODA) Langmuir monolayers was followed in time by monitoring the pressure-area isotherms. A large (and slow) expansion of the ODA monolayer was observed during immobilization of single-stranded DNA at the air-water interface, and interestingly, no further expansion was detected on addition of PNA to the subphase. However, quartz crystal microgravimetry and UV-melting analysis of Langmuir-Blodgett films of the ODA-PNA-DNA hybrid monolayer clearly showed that the sequential immobilization process had resulted in hybridization of the complementary single-stranded DNA molecules with PNA at the air-water interface.