Netrin 4 protein and its homologs are found throughout the animal kingdom. Netrin-4 is known to have a protective role against vascular damage. Previous studies have shown that human netrin-1 has a role in angiogenesis. This information about human netrin-1 and netrin-4 led us to research the pathway of netrin-4 in Tetrahymena thermophila. Our previous studies of the netrin proteins show that netrin-1 and netrin-3 are both repellents in Tetrahymena thermophila. The data in this study show that netrin-4 is also a repellent of Tetrahymena thermophila. These data suggest that netrin-4 could signal through the same pathway as netrin-1 and netrin-3. Furthermore, in our previous studies of the signaling pathways for netrin-1 and netrin-3, we discovered that netrin-1 signals through a tyrosine kinase pathway and netrin-3 signals through a serine/threonine kinase pathway. Because of this we decided to investigate whether or not netrin-4 signals through the same or different pathway as netrin-1 or netrin-3. We performed a cross adaptation assay on Tetrahymena thermophila using netrin-1, netrin-3, and netrin-4. The data from this assay suggests that netrin-4 does not signal through the same pathway as netrin-1 or netrin-3. Netrin-4 also was shown to have no effect on mitosis, which is similar to previous findings with netrin-1 in this organism. In contrast, netrin-3 peptides have been shown to inhibit mitosis in this organism.