<p><strong>Abstract.</strong> Molecular fossils, like bacterial branched glycerol dialkyl glycerol tetraethers (brGDGTs), and the stable isotopic composition of biomarkers, such as &#948;<sup>2</sup>H of leaf wax-derived <i>n</i>-alkanes (&#948;<sup>2</sup>H<sub><i>n</i>-alkane</sub>) or &#948;<sup>18</sup>O of hemicellulose-derived sugars (&#948;<sup>18</sup>O<sub>sugar</sub>) are increasingly used for the reconstruction of past climate and environmental conditions. Plant-derived &#948;<sup>2</sup>H<sub><i>n</i>-alkane</sub> and &#948;<sup>18</sup>Osugar values record the isotopic composition of plant source water (&#948;<sup>2</sup>H/&#948;<sup>18</sup>O<sub>source-water</sub>), which usually reflects mean annual precipitation (&#948;<sup>2</sup>H/&#948;<sup>18</sup>O<sub>precipiation</sub>), modulated by evapotranspirative leaf water enrichment and biosynthetic fractionation. Accuracy and precision of respective proxies should be ideally evaluated at a regional scale. For this study, we analysed topsoils below coniferous and deciduous forests, as well as grassland soils along a Central European transect in order to investigate the variability and robustness of various proxies, and to identify effects related to vegetation. Soil pH-values derived from brGDGTs correlate reasonably well with measured soil pH-values, but systematically overestimate them (&#916;pH&#8201;=&#8201;0.6&#8201;&#177;&#8201;0.6). The branched vs. isoprenoid tetraether index (BIT) can give some indication whether the pH reconstruction is reliable. Temperatures derived from brGDGTs overestimate mean annual air temperatures slightly (&#8710;TMA&#8201;=&#8201;0.5&#8201;&#176;C&#8201;&#177;&#8201;2.4). Apparent isotopic fractionation (&#949;<sub><i>n</i>-alkane/precipitation</sub> and &#949;<sub>sugar/precipitation</sub>) is lower for grassland sites than for forest sites due to &quot;signal damping&quot;, i.e. grass biomarkers do not record the full evapotranspirative leaf water enrichment. Coupling &#948;<sup>2</sup>H<sub><i>n</i>-alkane</sub> with &#948;<sup>18</sup>O<sub>sugar</sub> allows to reconstruct the stable isotopic composition of the source water more accurately than without the coupled approach (&#916;&#948;<sup>2</sup>H&#8201;=&#8201;~-21&#8201;&#8240;&#8201;&#177;&#8201;22 and &#916;&#948;<sup>18</sup>O&#8201;=&#8201;~-2.9&#8201;&#8240;&#8201;&#177;&#8201;2.8). Similarly, relative humidity during daytime and vegetation period (RHMDV) can be reconstructed using the coupled isotope approach (&#916;RHMDV&#8201;=&#8201;~-17&#8201;&#177;&#8201;12). Especially for coniferous sites, reconstructed RHMDV values as well as source water isotope composition underestimate the measured values. This can be likely explained by understory grass vegetation at the coniferous sites contributing significantly to the <i>n</i>-alkane pool but only marginally to the sugar pool in the topsoil. The large uncertainty likely reflect the fact that biosynthetic fractionation is not constant, as well as microclimate variability. Overall, GDGTs and the coupled &#948;<sup>2</sup>H<sub><i>n</i>-alkane</sub>-&#948;<sup>18</sup>O<sub>sugar</sub> approach have great potential for more quantitative paleoclimate reconstructions.</p>