We have previously shown that human monocytes express HILDA/LIF gene and its protein product upon stimulation. In the present study we have investigated some of the possible mechanisms involved in the regulation of HILDA/LIF gene expression in activated human monocytes and compared them to those of granulocyte colony-stimulating factor (G-CSF) and IL-1 beta gene expression. In the absence of added stimuli HILDA/LIF mRNA was barely detectable in isolated monocytes. HILDA/LIF mRNA accumulation was weakly induced by stimuli such as LPS or phorbol ester. However, a synergy was observed when each stimulus was used in combination with 1,25-dihydroxyvitamin D3. Nuclear run-on analysis did not detect an increase in HILDA/LIF gene transcription upon stimulation with LPS or phorbol ester in combination with 1,25-dihydroxyvitamin D3. HILDA/LIF mRNA half-life showed an increase when phorbol ester and 1,25-dihydroxyvitamin D3 were used in combination over that obtained for each stimuli alone. HILDA/LIF mRNA expression was largely inhibited when monocytes were stimulated in the presence of cycloheximide (CHX) added either at the onset or 4 h after the beginning of the stimulation period. When CHX was added at later time points, 2 h before cell harvesting, HILDA/LIF mRNA levels were superinduced when compared with those in cells stimulated in the absence of CHX. This superinduction can be at least partially explained by post-transcriptional mechanisms, since HILDA/LIF mRNA half-life in CHX-superinduced cells was significantly increased when compared with values on stimulated CHX-untreated cells. In contrast to HILDA/LIF, G-CSF and IL-1 beta mRNA accumulation showed a different response pattern to the same stimuli, and a augmentation of their gene transcription was detected by the run-on technique in activated monocytic cells when compared with controls. Furthermore, G-CSF and Il-1 beta mRNA levels were superinduced in monocytes stimulated in the presence of CHX from the onset of the stimulation period. These studies indicate that, when compared to G-CSF and IL-1 beta, the expression of HILDA/LIF gene is under the control of relatively specific regulatory mechanisms. HILDA/LIF gene expression is controlled by newly synthesized proteins acting at early and late time points of the stimulation period and with opposite effects on HILDA/LIF mRNA levels. Finally, HILDA/LIF mRNA levels are regulated by post-transcriptional mechanisms of mRNA stabilization probably controlled through labile newly synthesized proteins.