Cell density has been implicated in the regulation of neuronal gene phenotype. This study tested the interaction of signal transduction pathways and the expression of tyrosine hydroxylase (TH) mRNA with varying cell density. Increasing cell density in a parental, wild type PC12 cell line elevated steady state levels of TH mRNA. Three observations suggested that this induction is not related to the cyclic AMP dependent signalling pathway: (1) Forskolin stimulated the level of TH mRNA similarly at multiple densities. (2) PKA deficient mutant PC12 cell lines that have either one third (A123.7, AB11) or 3% (A126-1B2) of normal basal expression of TH mRNA still exhibit the same density induced elevation of TH mRNA levels as the wild type. (3) Different cell densities did not change cyclic AMP concentrations in the basal or in the receptor stimulated state. Increasing cell density did not change basal levels of inositol triphosphate (IP3) levels, which suggests that the phosphatidylinositol cascade (PI) is not responsible for density dependent changes in TH expression. Increasing confluence was highly correlated to [Ca2+]i in control (r = 0.70; P < 0.0001), A123.7 (r = 0.92; P < 0.001), AB11 (r = 0.72; P < 0.0001) and A126 (r = 0.42; P < 0.07). Taken together, the results show that neither cyclic AMP nor the PI cascade is involved in cell density induced changes in TH mRNA and suggest that altered [Ca2+]i may have a role.