The molecular mechanism of the anti-inflammatory effect of erythromycin (EM) was investigated at the level of transcriptional regulation of cytokine gene expression in T cells. EM (>10(-6) M) significantly inhibited interleukin-8 (IL-8) expression but not IL-2 expression from T cells induced with 20 ng of phorbol 12-myristate 13-acetate (PMA) per ml plus 2 microM calcium ionophore (P-I). In electrophoretic mobility shift assays EM at 10(-7) to 10(-5) M concentrations inhibited nuclear factor kappa B (NF-kappaB) DNA-binding activities induced by P-I. Reporter gene assays also showed that EM (10(-5) M) inhibited IL-8 NF-kappaB transcription by 37%. The inhibitory effects of EM on transcriptional activation of IL-2 and DNA-binding activity of nuclear factor of activated T cells (NFAT) were not seen in T cells. On the other hand, FK506, which is also a macrolide derivative, inhibited transcriptional activation of both NF-kappaB and NFAT more strongly than EM did. The mechanism of EM inhibition of transactivation of NF-kappaB was further investigated in transiently transfected T cells that express calcineurin A and B subunits. Expression of calcineurin did not render transactivation of NF-kappaB in T cells more resistant to EM, while the inhibitory effect of FK506 on transactivation of NF-kappaB was attenuated. These findings indicate that EM is capable of inhibiting expression of the IL-8 gene in T cells through transcriptional inhibition and that this inhibition is mediated through a non-calcineurin-dependent signaling event in T lymphocytes.