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3-Hydroxy fatty acids (3-OH-FAs), derived from Gram-negative bacterial outer membranes, have received recent attention for their potential as new terrestrial pH and temperature proxies for palaeoclimate studies. Initial studies from altitudinal transects of contemporary soils – correlating bacterial 3-OH-FA compositions to air temperature and pH – have shown promising results. But the geographical extent of recent calibrations is limited. In this study, we analyse 3-OH-FA lipid distributions in 186 globally distributed soil samples to study the environmental factors controlling the relative distribution of the 3-OH-FA isomers. Our sample-set covers a wide range of temperatures (−0.4 to 27 °C) and pH (3.6–9.2). For the global compilation we find that the ratio of anteiso to normal 3-OH-FAs of the C₁₅ or C₁₇ homologues (RAN₁₅ or RAN₁₇) shows a strong linear relationship with mean annual air temperature (MAAT) (R² …