alpha 1-Adrenoceptors (ARs) comprise a heterogeneous family, and subtype-selective ligands are valuable for studying the functional role of each receptor subtype. We characterized a newly synthesized, alpha 1-AR antagonist, KMD-3213, by using Chinese hamster ovary cells stably expressing the three cloned human alpha 1-ARs (alpha 1a, alpha 1b, and alpha 1d), as well as native rat and human tissues. KMD-3213 potently inhibited 2-[2-(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl]-alpha-tetralone binding to the cloned human alpha 1a-AR, with a Ki value of 0.036 nM, but had 583- and 56-fold lower potency at the alpha 1b- and alpha 1d-ARs, respectively. KMD-3213 inhibited norepinephrine-induced increases in intracellular Ca2+ concentrations in alpha 1a-AR-expressing Chinese hamster ovary cells with an IC50 of 0.32 nM but had a much weaker inhibitory effect on the alpha 1b- and alpha 1d-ARs. Using pharmacologically well characterized native rat tissues [submaxillary gland (alpha 1A-AR-expressing tissue), liver (alpha 1B-AR-expressing tissue), and heart (mixed alpha 1A- and alpha 1B-AR-expressing tissue)], binding studies showed that inhibition curves for KMD-3213 in submaxillary gland and liver best fit a one-site model (with Ki values of 0.15 and 16 nM, respectively), whereas KMD-3213 had high and low affinity sites in heart membranes. Chloroethylclonidine treatment of rat heart membranes completely eliminated the low affinity sites for KMD-3213. Furthermore, in human liver and prostate KMD-3213 could identify high and low affinity sites, the Ki values of which corresponded well to those for the cloned human alpha 1a- and alpha 1b-ARs, respectively. Moreover, the affinity of KMD-3213 was found to be approximately 10-fold higher at the cloned human alpha 1a-AR than at the cloned rat alpha 1a-AR. KMD-3213 is a potent and highly selective antagonist for the human alpha 1a-AR and would be useful for studying the physiological roles of human alpha 1-AR subtypes.