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In early work on coumarin in sweetclover, the assumption was made that the free form of the compound predominated in the intact plant. Subsequent investigations demonstrated the presence of bound coumarin in addition to free, and it was then thought that both forms were normally present. Recent work indicates that when suitable extraction procedures are used, virtually all the coumarin is obtained in the bound form. Such extraction procedures must provide for the rapid inactivation of β-glucosidase, thus preventing the hydrolysis of bound coumarin (apparently the glucoside of cis-o-hydroxycinnamic acid) during the extraction process. In the present study, inactivation of β-glucosidase was achieved by submersion of the plant tissue in boiling water. When this step was incorporated into the extraction procedure, leaf samples of 4 homozygous genotypes (cucubb, cucuBB, CuCubb, and CuCuBB) and 9 coumarin-containing varieties of sweetclover, and of sweet vernal grass and sweet grass were found to be essentially devoid of free coumarin. In general, any treatment that caused disruption of cellular or tissue organization prior to inactivation of β-glucosidase resulted in conversion of bound coumarin to the free form. Therefore, care in handling the plant tissue is essential. Implications of the apparent absence of free coumarin in the intact sweetclover plant are discussed with respect to the possible roles of the B/b alleles and β-glucosidase in coumarin biosynthesis.