We examined the effectiveness of bovine cholesterol concentrate in reducing the high level (10-20%) of fetal bovine serum (FBS) necessary to promote tick cell growth in vitro. Tick cell lines isolated from embryos of Anocentor nitens (ANE 58), Boophilus microplus (BME 26), and Rhipicephalus appendiculatus (RAE 25) were used. They were incubated in L-15 (BME 26) or L-15B (ANE 58 and RAE 25) supplemented with 10% tryptose phosphate broth (TPB), 5% (ANE 58 and BME 26) or 3% FBS, 10-90 microns/ml cholesterol. A concentration of 10 micrograms/ml cholesterol stimulated the growth rate of all three lines but more than 30 micrograms/ml depressed growth in ANE 58 and RAE 25 cells, while multiplication of BME 26 cells was enhanced by all cholesterol concentrations tested. All three lines could be continuously grown in 5% FBS, provided that 10 micrograms/ml cholesterol was included. Nutrients added to L-15 in the formulation of L-15B were tested singly or in combination for their ability to support tick cell growth in medium supplemented only with 5% FBS and 10 microns/ml cholesterol. In L-15 alone, RAE 25 cells did not multiply. Adding glucose (Glc), glutamic acid (Glu), or alpha-ketoglutaric acid (alpha K) had little or no effect, and the same was true for combinations of Glc plus alpha K, aspartic acid (Asp) plus proline (Pro) and glutamine (Gln), and minerals plus vitamins (MV). When Asp, Gln, Pro, and alpha K were combined with Glc and/or MV and added to L-15, there was appreciable growth stimulation, but best results were obtained when Glu was also included. In this medium, i.e., L-15B with 5% FBS and 10 mu/ml cholesterol, lines BME 26 and RAE 25 could be continuously subcultured.