Accurate fixation is a must for the assessment of myelinated nerve fiber morphology and transcardial perfusion and immersion methods are the most commonly used fixation techniques. In the present study we designed a new fixation technique for the histomorphometric and stereological evaluation of sciatic nerve fiber and referred it as instillation fixation. The method involved preliminary in situ fixation of the nerve sample without dissecting it from the animal body followed by a complete conventional fixation protocol. The objective of this study was to compare the three fixation techniques with each other for fixation artifacts. Eighteen female Wistar albino rats constituted the study material. The animals were allocated into three experimental groups corresponding to different fixation methods (immersion, instillation and transcardial perfusion, respectively). Quantitative assessments of nerve samples harvested from the animals of each group included the number of total myelinated axons, normal myelinated axons, alterations in myelin compaction, myelinated axons with irregular fiber shape, and with myelin loops and g ratio. Results revealed that normal nnyelinated axons were markedly lower in the immersion fixation group compared to those of instillation and transcardial perfusion. Moreover a significant decrease was noted with respect to alterations in myelin compaction in the instillation fixation group. In contrast, no significant difference was observed in myelin thickness and axon cross sectional area. In conclusion instillation fixation technique proved to be a valid and simple method for the assessment of peripheral nerve morphology for further analyses in a rat model.