The biosynthesis of polyamines from the diamine putrescine is not fully understood in higher plants. A putrescine aminopropyltransferase (PAPT) enzyme activity was characterized in alfalfa (Medicago sativa L.). This enzyme activity was highly specific for putrescine as the initial substrate and did not recognize another common diamine, 1,3-diaminopropane, or higher-molecular-weight polyamines such as spermidine and spermine as alternative initial substrates. The enzyme activity was inhibited by a general inhibitor of aminopropyltransferases, 5'-methylthioadenosine, and by a specific inhibitor of PAPTs, cyclohexylammonium sulfate. The initial substrate specificity and inhibition characteristics of the enzyme activity suggested that it is a classical example of a PAPT. However, this enzyme activity yielded multiple polyamine products, which is uncharacteristic of PAPTs. l h e major reaction product of PAPT activity i n alfalfa was spermidine. The next most abundant products of the enzyme reaction using putrescine as the initial substrate included the tetramines spermine and thermospermine. These two tetramines were distinguished by thin-layer chromatography to be distinct reaction products exhibiting differential rates of formation. In addition, the uncommon polyamines homocaldopentamine and homocaldohexamine were tentatively identified as minor enzymatic reaction products but only in extracts prepared from osmotic stresstolerant alfalfa cultivars. PAPT activity from alfalfa was highest i n meristematic shoot tip and floral bud tissues and was not detected in older, nonmeristematic tissues. Product inhibition of the enzyme activity was observed after spermidine was added into the i n vitro assay for alfalfa PAPT activity. A biosynthetic pathway is proposed that accounts for the characteristics of this PAPT activity and accommodates a nove1 scheme by which certain uncommon polyamines are produced in plants.The diamine putrescine and the polyamines spermidine and spermine are ubiquitous components of living organisms and are essential primary metabolites for normal growth and development (Flores et al., 1989). These three compounds are often referred to as common polyamines, and polyamines with a restricted natural occurrence may