Four patients with juvenile neuronal ceroid lipofuscinoses, a childhood neurodegenerative disorder that was previously described as CLN9 variant, are reclassified as CLN5 disease. CLN5-deficient (CLN5-/-) fibroblasts demonstrate adhesion defects, increased growth, apoptosis, and decreased levels of ceramide, sphingomyelin, and glycosphingolipids. The CLN8 protein (CLN8p) corrects growth and apoptosis in CLN5-/- cells. Related proteins containing a Lag1 motif (CerS1/2/4/5/6) partially corrected these deficits, with CerS1, which is primarily expressed in brain, providing the best complementation, suggesting CLN5p activates CerS1 and may co-immunoprecipitate with it. CLN8p complements CLN5-deficient cells, consolidating the interrelationship of CLN5p/CLN8p, whose potential roles are explored as activators of (dihydro)ceramide synthases. Homozygosity mapping using microarray technology led to identification of CLN5 as the culprit gene in previously classified CLN9-defective cases. Similar to CLN5-/-cells, ceramide synthase activity, C16/C18:0/C24:0/C24:1 ceramide species, measured by MS is decreased in CLN8-/- cells. Comparison of normal versus CLN5-/- cell CerS1-bound proteins by immunoprecipitation, differential gel electrophoresis, and MS revealed absence of ?-actin in CLN5-/- cells. The ?-actin gene sequence is normal in CLN5-/- derived DNA. The ?-actin-bound proteins, vimentin and histones H2Afz/H3F3A/Hist1H4, were absent from the ?-actin protein complex in CLN5-/- cells. The function of CLN5p may require vimentin and the histone proteins to bind ?-actin. Defective binding could explain the CLN5-/- cellular phenotype. We explore the role of the CLN5/CLN8 proteins in ceramide species specific sphingolipid de novo synthesis, and suggest that CLN5/CLN8 proteins are more closely related than previously believed.