This paper combines new experimental data for electrokinetic characterization of hydrophobic polymers with a detailed discussion of the putative origins of charge at water-hydrophobe interfaces. Complexities in determining the origin of charge are discussed in the context of design and modeling challenges for electrokinetic actuation in hydrophobic microfluidic devices with aqueous working fluids. Measurements of interfacial charge are complicated by slip and interfacial water structuring phenomena (see Part 2, this issue). Despite these complexities, it is shown that (i) several hydrophobic materials, such as Teflon and Zeonor, have predictable electrokinetic properties and (ii) electrokinetic data for hydrophobic microfluidic systems is most consistent with the postulate that hydroxyl ion adsorption is the origin of charge.