Fluctuations in ionized cytosolic calcium ([Ca2+]i) are considered important signals for induction of growth or differentiation in mammalian cells. The resting concentrations of [Ca2+]i in normal and adenovirus 12-SV40 hybrid virus-transformed (BEAS-2B) human bronchial epithelial cells were 63 +/- 15 nM (SD) and 44 +/- 15 nM, respectively. Eight % calcium-free fetal bovine serum rapidly caused a significant increase in [Ca2+]i, while causing both cell types to undergo squamous differentiation. When treated with 8% calcium-free fetal bovine serum, a serum-sensitive subclone of BEAS-2B cells exhibited a higher elevation of [Ca2+]i than a serum-resistant (i.e., not stimulated to differentiate by serum) subclone. However, a serum component involved in the induction of squamous differentiation, transforming growth factor type beta, did not increase [Ca2+]i in either normal cells or BEAS-2B cells. 12-O-Tetradecanoyl-phorbol-13-acetate, an exogenous inducer of squamous differentiation and activator of protein kinase C, did not increase [Ca2+]i, but did attenuate serum-induced elevation of [Ca2+]i. These results suggest that while an increase in [Ca2+]i is associated with serum-induced squamous differentiation, a cytosolic ionized calcium signal is not required for the initiation of the squamous differentiation pathway induced by either transforming growth factor type beta or 12-O-tetradecanoylphorbol-13-acetate.