An improved guinea pig headholder, designed to provide long-term and reliable head fixation, was developed. It consists of a mouthpiece, nose clamp, and pivoting backing block. The advantages of this headholder are i) the correspondence of the mouthpiece to the anatomical peculiarities of the guinea pig skull, ii) automatic positioning for snout fixation, and iii) application of the load to sites of the skull that are strong, the latter of which prevents tissue damage. The headholder fits guinea pigs weighing 400-750 g and permits free access to all brain regions.