We have used a reversible permeabilization method to facilitate the entry of Giα1, 2 and 3 G‐protein subunit mRNA antisense or mismatch oligonucleotides into intact tissue, to investigate the G‐protein α‐subunit coupling of α2‐adrenoceptors, neuropeptide Y (NPY) Y1, and A1 adenosine receptors in preparations of the epididymis of the guinea‐pig. The α2‐adrenoceptor agonist, xylazine, elicited concentration dependent contractions from preparations of phenylephrine (3 μM)‐stimulated epididymis (pEC50 value 6.52±0.39, maximum response 236±41 mg force). Compared to respective mismatch controls the incubation of preparations with Giα2, but not with Giα1 or Giα3 mRNA antisense oligonucleotides (30 μM) reduced the maximal xylazine‐potentiation of phenylephrine (3 μM)‐stimulated contractility (to 51±12% of Giα2 mismatch control). The oligonucleotide incubations had no effect upon the pEC50 values of xylazine. The A1 adenosine receptor agonist, cyclopentyladenosine (CPA) elicited concentration dependent contractions from preparations of phenylephrine (3 μM)‐stimulated epididymis (pEC50 value 7.66±0.57, maximum response 208±54 mg force). Incubation of preparations of epididymis with Giα1, but neither Giα2 nor Giα3 antisense oligonucleotides reduced the maximal CPA‐potentiation of phenylephrine (3 μM)‐stimulated contractions (to 55±17% of Giα1 mismatch control), pEC50 values were not affected. The incubation of preparations with Giα2 antisense mRNA oligonucleotides reduced the maximal NPY‐potentiation of phenylephrine (3 μM)‐stimulated contractions (to 62±15% of Giα mismatch control). Compared with Giα2 mismatch controls, the incubation of preparations with Giα1 and Giα3 oligonucleotides also reduced the NPY‐potentiation of phenylephrine (3 μM)‐stimulated contractions. These studies indicate that, in the guinea‐pig epididymis, α2‐adrenoceptors and A1 adenosine receptors preferentially couple to effectors through Giα2 and Giα1 subunits respectively. In contrast NPY receptors may elicit effects through either Giα1, 2 or 3 subunits. British Journal of Pharmacology (1999) 127, 85–90; doi:10.1038/sj.bjp.0702515