Infections with Eimeria ninakohlyakimovae represent important coccidian diseases of goats severely affecting animal health and profitability of goat industry. For the development of suitable vaccination strategies basic research is needed for which one important prerequisite is the establishment of in vitro cultures guaranteeing the availability of parasitic material. Therefore, primary cell cultures [caprine, bovine and human umbilical vein endothelial cells (CUVEC, BUVEC, HUVEC)] as well as permanent cell lines [bovine foetal gastrointestinal cells (BFGC), bovine colonic epithelial cells (BCEC), African green monkey kidney cells (VERO)] were exposed to vital sporozoites of E. ninakohlyakimovae. The parasites invaded all different cell types used, irrespective of their origin, but further development into macromeronts and subsequent release of viable merozoites I were restricted to ruminant cells. Mature macromeronts developed in both, endothelial (CUVEC, BUVEC) and epithelial cells (BCEC). VERO cells were non-permissive for parasite development, nevertheless sporozoites survived for 21 days p.i. within an enlarged parasitophorous vacuole. Best in vitro development of E. ninakohlyakimovae macromeronts with respect to the production of viable merozoites I was observed in BCEC, followed by BUVEC. However, the largest macromeronts developed in CUVEC. Mature macromeronts were also detected in BFGC, but these cells were less effective concerning infection rates and productivity. The complete life-cycle of E. ninakohlyakimovae leading to oocyst production was not accomplished in any cell type used. In conclusion, we established suitable in vitro systems for the culture of E. ninakohlyakimovae macromeronts, e.g., for the mass production of merozoites I, for basic studies on parasite/host endothelial cell interactions or for pharmaceutical screenings.