Previous data have demonstrated that CXC-chemokine platelet factor 4 (PF4) inhibits the proliferation of the human erythroleukemia cell line (HEL). However, the mechanism of action is unclear at present. The signaling transduction induced by PF4 in the HEL was compared with that induced by transforming growth factor beta1 (TGF-beta1), which is also a potent inhibitor of HEL growth. It was found that PF4 had no inhibitory effect on intracellular calcium levels in resting HEL cells. When HEL cells were stimulated with interleukin-3 (IL-3), a rapid increase in the intracellular level of free calcium occurred within 15 to 20 seconds, and this increase was followed by a sustained increase that gradually declined until resting levels were reached 30 to 40 minutes later. PF4 dramatically decreased the transient rise of [Ca2+] and protein kinase C (PKC) activity of HEL cells induced by IL-3. However, PF4 had no inhibitory effect on PKC activation in resting HEL cells. Furthermore, PF4 was found to down-regulate significantly protein tyrosine kinase (PTK) activity. In contrast, TGF-beta1 induced an increase in intracellular free calcium concentration and PKC and PTK activity in HEL cells. Furthermore, PF4 significantly increased the messenger RNA (mRNA) level of p21waf1 in HEL cells. These data demonstrate that PF4 acts on HEL cells through a signaling transduction pathway, which is different from that of TGF-beta1 and is related to the up-regulatory mRNA level of p21waf1 in HEL cells.