The purpose of the study was to evaluate the role which non-essential envelope glycoproteins play in the neuroinvasion and neural spread of ADV. The invasion and spread in the trigeminal nervous pathway with the Ka strain of ADV and its single deletion mutants Ka gI-, Ka gp63- and Ka gIII- were examined after intranasal inoculation in neonatal pigs by virus isolation and immunocytochemistry. Evaluation was performed in the nasal mucosa, trigeminal ganglion (1st neuronal level), ponsmedulla (2nd neuronal level) and thalamus-cerebellum (3rd neuronal level). The Ka gIII- mutant invaded up to the 3rd neuronal level of the trigeminal pathway and spread in a similar way to the parental Ka strain. The Ka gp63- mutant invaded up to the 3rd neuronal level but the spread of this mutant was impaired at all the neuronal levels. The Ka gI- mutant was least neuroinvasive and reached only up to the 2nd neuronal level. The results showed that glycoproteins gI and gp63 play a role in the invasion and spread of ADV in the nervous system. However, the gI glycoprotein appears to be the most important for neuroinvasion and neural spread of ADV in pigs. Therefore, gI deleted vaccines may be considered to be safer with respect to the neuroinvasion than vaccines carrying single deletions of other non-essential envelope glycoproteins.