The role of accessory abducens nucleus neurons in the conditioned eye retraction/nictitating membrane extension response was defined in the rabbit. Horseradish peroxidase injections into the retractor bulbi muscle showed that accessory abducens nucleus is the principal location of its motorneurons. Single and multiple unit recording in accessory abducens indicated that these motor neurons show a marked responsiveness to cornea1 and periorbital stimulation and fire in close correlation with conditioned, unconditioned, or spontaneous eye retraction/nictitating membrane extension. Complete lesions of accessory abducens showed, at most, a partial reduction of the conditioned and unconditioned eye retraction response. Section of the extraocular muscles, other than retractor bulbi, also caused a partial reduction of the eye retraction response. Accessory abducens lesions, combined with extraocular muscle section, were necessary to dramatically reduce the eye retraction response permanently. These experiments demonstrated that accessory abducens is a primary controller of eye retraction through its axons to retractor bulbi. The other extraocular muscles act in concert with retractor bulbi to elicit conditioned and unconditioned eye retractions.Analysis of the neural substrates of associative learning in mammalian brain is a complex problem. The use of a relatively simple behavioral paradigm in which the stimuli to be associated are easily controlled, in which at least one behavioral output is readily measured, and in which physiological recordings may be made in the animal during and after learning will facilitate progress in understanding the learning process. The rabbit nictitating membrane (NM) preparation, which was originally described by Gormezano et al. (1962) has many characteristics making it desirable for use in physiological and anatomical studies of learning (Thompson, 1976; Disterhoft et al., 1977). It and closely related eyeblink conditioning (Woody and Brozek, 1969) have been adapted by several groups as "model systems" in which to study the systems neurophysiology of learning.A typical approach is to pair an auditory conditioned stimulus (CS) with a cornea1 air puff or periorbital shock unconditioned stimulus