The microwave spectrum for o-benzyne was obtained by passing a dilute (<1%) mixture of benzene in neon through a pulsed-DC discharge nozzle source into a pulsed-beam, Fourier transform spectrometer. Rotational transitions were measured for the normal isotopomer, the two unique single-D isotopomers, and the C-13 isotopomer and one of the C-13 isotopomers. Benzynes have been known as reactive intermediates in organic reactions for many years, and have recently been implicated in gasoline combustion reactions and antitumor activity of enediynes. Twenty-seven b-type transitions for the normal isotopomer were fit to obtain A=6989.7292(6), B=5706.8062(6), and C=3140.3708(4) MHz, and five centrifugal distortion constants. The inertial defect is Delta=0.069 4 amu A(2), consistent with a planar structure. Hyperfine structure for the D-1 (closest to the CdropC bond) and D-2 (furthest from the CdropC bond), was analyzed to obtain deuterium quadrupole coupling strengths eQq(aa)(D-1)=185(3) kHz, eQq(bb)(D-1)=-85(2) kHz, eQq(aa)(D-2)=5(13), and eQq(bb)(D-2)=86(13) kHz. The C-D, bond axis quadrupole coupling strengths are compared with values for benzene. Spectra for the C-13(6) and one of the C-13(1) isotopomers were analyzed to obtain rotational constants. Kraitchman analysis and least-squares fitting provided nearly all of the structural parameters. The preliminary structural analysis yields an acetylenic Cequivalent toC bond length of 1.24 A, in agreement with a recent NMR value. Density functional theory (DFT) calculations were used to obtain structural parameters, and values obtained are in very good agreement with present experimental results. (C) 2003 American Institute of Physics.