In situ-formed cobalt(I) complexes are proposed to act as efficient catalysts in regioselective Diels-Alder reactions of unactivated substrates such as 1,3-dienes and alkynes. We report the first experimental evidence for the in situ reduction of CoBr2(dppe) [dppe = 1,2-bis(diphenylphosphino)ethane] by Zn/ZnI2 to [Co(I)(dppe)](+) by means of electrospray MS(n) experiments. Additionally, the reactivities of Co(II) and Co(I) dppe complexes toward the Diels-Alder substrates isoprene and phenylacetylene were probed in gas-phase ion/molecule reactions (IMRs). Isoprene and phenylacetylene were introduced into the mass spectrometer via the buffer gas flow of a linear ion trap. The IMR experiments revealed a significantly higher substrate affinity of [Co(I)(dppe)](+) compared with [Co(II)Br(dppe)](+). Furthermore, the central intermediate of the solution-phase cobalt-catalyzed Diels-Alder reaction, [Co(I)(dppe)(isoprene)(phenylacetylene)](+), could be generated via IMR and examined in the gas phase. Collision activation of this complex ion delivered evidence for the gas-phase reaction of isoprene with phenylacetylene in the coordination sphere of the cobalt ion. The experimental findings are consistent with the results of quantum-chemical calculations on all of the observed Co(I) dppe complex ions. The results constitute strong analytical evidence for the formation and importance of different cobalt(I) species in regioselective Diels-Alder reactions of unactivated substrates and identify [Co(I)(dppe)](+) as the active Diels-Alder catalyst.