Phospholipid monolayers formed at oil-water interfaces are used for various biological applications. However, monolayer structures are not well understood. Herein, we investigated hydrocarbon partitioning in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine monolayers formed at hydrocarbon-water interfaces using fluorescence microscopy and pendant drop tensiometry. The monolayers strongly interacted with squalene, n-hexadecane, n-tetradecane, n-dodecane, n-decane, and n-butylcyclohexane. These alkane and alkylcyclohexane molecules remained within the monolayers during area compression. In contrast, the monolayers interacted weakly with n-pentylbenzene and n-butylbenzene. These alkylbenzenes were gradually removed from the monolayers upon area compression and were completely expelled at an area per lipid of ∼70 Å2. Surface pressure analysis indicated that the ability of hydrocarbons to penetrate the monolayers was enhanced in the order of n-butylbenzene < n-pentylbenzene < n-butylcyclohexane < n-hexadecane.