Stable isotope ratios (δ13C and δ18O) of tree-ring α-cellulose are important tools in paleoclimatology, ecology, plant physiology and genetics. The Multiple Sample Isolation System for Solids (MSISS) was a major advance in the tree-ring α-cellulose extraction methods, offering greater throughput and reduced labor input compared to traditional alternatives. However, the usability of the method for resinous conifer species may be limited by the need to remove extractives from some conifer species in a separate pretreatment step. Here we test the necessity of pretreatment for α-cellulose extraction in loblolly pine (Pinus taeda L.), and the efficiency of a modified acetone-based ambient-temperature step for the removal of extractives (i) in loblolly pine from five geographic locations representing its natural range in the southeastern USA, and (ii) on five other common coniferous species (black spruce (Picea mariana Mill.), Fraser fir (Abies fraseri (Pursh) Poir.), Douglas fir (Pseudotsuga menziesii (Mirb.) Franco), Norway spruce (Picea abies (L.) Karst) and ponderosa pine (Pinus ponderosa D.)) with contrasting extractive profiles. The differences of δ13C values between the new and traditional pretreatment methods were within the precision of the isotope ratio mass spectrometry method used (±0.2‰), and the differences between δ18O values were not statistically significant. Although some unanticipated results were observed in Fraser fir, the new ambient-temperature technique was deemed as effective as the more labor-consuming and toxic traditional pretreatment protocol. The proposed technique requires a separate acetone-inert multiport system similar to MSISS, and the execution of both pretreatment and main extraction steps allows for simultaneous treatment of up to several hundred microsamples from resinous softwood, while the need of additional labor input remains minimal.Read less <