Heme functions not only as a prosthetic group of hemoproteins but also as a regulatory molecule, suggesting the presence of 'free' heme. Classically, total non-covalently bound heme is extracted from plant samples with acidic acetone after removal of pigments with basic and neutral acetone. Earlier work proposed that free heme can be selectively extracted into basic acetone. Using authentic hemoproteins, we confirmed that acidic acetone can quantitatively extract heme, while no heme was extracted into neutral acetone. Meanwhile, a certain amount of heme was extracted into basic acetone from hemoglobin and myoglobin. Moreover, basic acetone extracted loosely bound heme from bovine serum albumin, implying that the nature of hemoproteins largely influences heme extraction into basic acetone. Using a highly sensitive heme assay, we found that basic and neutral acetone can extract low levels of heme from plant samples. In addition, neutral acetone quantitatively extracted free heme when it was externally added to plant homogenates. Furthermore, the level of neutral acetone-extractable heme remained unchanged by precursor (5-aminolevulinic acid) feeding, while increased by norflurazon treatment which abolishes chloroplast biogenesis. However, changes in these heme levels did not correlate to genomes uncoupled phenotypes, suggesting that the level of unbound free heme would not affect retrograde signaling from plastids to the nucleus. The present data demonstrate that the combination of single-step acetone extraction following a sensitive heme assay is the ideal method for determining total and free heme in plants.