Most attempts to develop in vitro models of the blood–brain barrier (BBB) have resulted in models with low transendothelial electrical resistances (TEER), as compared to the native endothelium. The aim of the present study was to investigate the impact of culture pH and buffer concentration on paracellular tightness of an established in vitro model of the BBB consisting of bovine brain capillary endothelial cells (BCEC) co-cultured with rat astrocytes. BCEC and rat astrocytes were isolated and co-cultured using astrocyte-conditioned media with cAMP increasing agonists and dexamethasone. The co-culture had average TEER values from 261 ± 26 Ω cm² to 760 ± 46 Ω cm² dependent on BCEC isolation batches. Furthermore, mRNA of occludin, claudin-1, claudin-5, JAM-1, and ZO-1 were detected. Increased buffer concentration by addition of HEPES, MOPS, or TES to the media during differentiation increased the TEER up to 1,638 ± 256 Ω cm² independent of the type of buffer. This correlated with increased expression of claudin-5, while expression of the other tight junction proteins remained unchanged. Thus, we show for the first time that increased buffer capacity of the medium during differentiation significantly increases tightness of the BCEC/astrocyte in vitro BBB model. This regulation may be mediated by increased claudin-5 expression. The observations have practical implications for generating tighter BBB cell culture models, and may also have physiological implications, if similar sensitivity to pH-changes can be demonstrated in vivo.