A method for the preparation of an in vivo eyecup and a complex stimulating-sampling device are described; these are suitable for long-term parallel neurochemical and electrophysiological experiments on the rat retina without any additives into the eyecup. In this in vivo eyecup the extracellular microenvironment is under the normal homeostatic control of the vascular system; no continuous exchange of the eyecup fluid and no addition of glutamate is necessary to maintain stable retinal electric responses and amino acid concentrations. The eyecup viability was tested by monitoring the electroretinogram (ERG) and the amino acid contents of the eyecup fluid sampled from the preretinal space by means of microdialysis. After the initial increase the b-wave of the ERG changed by less than 10% in maximal amplitude during experiments lasting 5 h. The glutamate, glutamine, and glycine levels proved comparatively, whereas the taurine level rose continuously throughout the experimental protocol. Recovery of ERG was achieved following exposure to bright background illumination. Total exchange of the eyecup volume requires 20 min at a flow rate of 1 microl/min. The effect of L-AP4 on the ERG was successfully reproduced, which suggests the applicability of this in vivo eyecup for pharmacological experiments on the rat retina.