Central nervous system (CNS) disease is one of the most common extra-respiratory tract complication of influenza A virus infections. Remarkably, zoonotic H5N1 virus infections are more frequently associated with CNS disease than seasonal or pandemic viruses. Little is known about the interaction between influenza A viruses and cells of the CNS, and therefore it is currently unknown which viral factors are important for efficient replication. Here, we determined the replication kinetics of a seasonal, pandemic, zoonotic, and lab-adapted influenza A virus in human neuron-like (SK-N-SH), astrocyte-like (U87-MG) cells and primary mouse cortex neurons. In general, highly pathogenic avian influenza (HPAI) H5N1 virus replicated most efficiently in all cells which was associated with efficient attachment and infection. Seasonal H3N2 virus and to a lesser extent pandemic H1N1 replicated trypsin-dependent in SK-N-SH but not in U87-MG cells. In the absence of trypsin, only HPAI H5N1 and WSN viruses replicated. Removal of the multi-basic cleavage site (MBCS) from HPAI H5N1 virus attenuated, but did not abrogate replication. Taken together, we showed that the MBCS and to a lesser extent ability to attach and are important determinants for efficient replication of HPAI H5N1 virus in cells of the CNS. This suggests that both an alternative HA cleavage mechanism and preference for α-2,3 linked sialic acids allowing efficient attachment, contribute to the ability of influenza A viruses to replicate efficiently in cells of the CNS. This study further improves our knowledge on potential viral factors important for the neurotropic potential of influenza A viruses.IMPORTANCE Central nervous system (CNS) disease is one of the most common extra-respiratory tract complications of influenza A virus infections and frequency and severity differ between seasonal, pandemic and zoonotic viruses. However, little is known about the interaction of these viruses with cells of CNS. Differences among seasonal, pandemic and zoonotic viruses in replication efficacy and cleavability in CNS cells partially explain the higher frequency and severity of zoonotic viruses. Identifying important viral factors and detailed knowledge of the interaction between influenza virus and CNS cells are important to prevent and treat this potentially lethal CNS disease.