Contact angle analysis of cell surface hydrophobicity (CSH) describes the tendency of a water droplet to spread across a lawn of filtered bacterial cells. Colistin-induced disruption of the Gram-negative outer membrane necessitates hydrophobic contacts with lipopolysaccharide (LPS). We aimed to characterize the CSH of Acinetobacter baumannii using contact angles, to provide insight into the mechanism of colistin resistance. Contact angles were analysed for five paired colistin-susceptible and resistant Ac. baumannii strains. Drainage of the water droplet through bacterial layers was demonstrated to influence results. Consequently, measurements were performed 0.66 s after droplet deposition. Colistin-resistant cells exhibited lower contact angles (38.8 +/- 2.846.8 +/- 1.3 degrees) compared with their paired colistin-susceptible strains (40.7 +/- 3.048.0 +/- 1.4 degrees; anova; P < 0.05). Contact angles increased at stationary phase (50.3 +/- 2.961.5 +/- 2.5 degrees and 47.4 +/- 2.050.8 +/- 3.2 degrees, susceptible and resistant, respectively, anova; P < 0.05) and in response to colistin 32 mg l-1 exposure (44.5 +/- 1.550.6 +/- 2.8 degrees and 43.5 +/- 2.248.0 +/- 2.2 degrees, susceptible and resistant, respectively; anova; P < 0.05). Analysis of complemented strains constructed with an intact lpxA gene, or empty vector, highlighted the contribution of LPS to CSH. Compositional outer-membrane variations likely account for CSH differences between Ac. baumannii phenotypes, which influence the hydrophobic colistinbacterium interaction. Important insight into the mechanism of colistin resistance has been provided. Greater consideration of contact angle methodology is necessary to ensure accurate analyses are performed.