© 1999, National Academy of Sciences of Ukraine. All rights reserved. A novel ribosomal S6 kinase, termed p70S6 kinase β, has been recently identified. It is highly homologous to known p70/p85 S6 kinase (p70S6 kinase α) in catalytic, kinase extension and auto-inhibitory domains, but differs significantly in the regulatory N- and C-terminal regions. Here we report the kinetics of serum-induced activation of cytoplasmic isoforms of p70S6 kinase α and β (p70α2 and p70β2) in transiently transfected HEK293. Both kinases exhibit biphasic activation upon serum stimulation, but differ in the appearance of peaks of their S6 specific activity. We also found that p70α2 and p70β2, when overexpressed in HEK293 cells, are sensitive to the immunosuppressant rapamycin. However, p70β2 kinase is more sensitive to low concentrations of rapamycin, when compared with p70α2. At the same time, high concentrations of rapamycin are inhibitory to a higher extent towards p70α2, than p70β2 kinase. Taken together, our results indicate that p70α2 kinase is activated earlier in response to serum and is less sensitive to high concentration of rapamycin, in comparison to p70α2. These differences indicate the existence of alternative signalling mechanisms, involved in the regulation of p70β2 kinase.