Melanoma growth stimulatory activity (MGSA)/growth regulated (GRO) and interleukin-8 (IL-8) are highly related chemokines that have a causal role in melanoma progression. Expression of these chemokines is similar in that both require the NF-kappa B element and additional regions such as the CAAT/enhancer binding protein (C/EBP) element of the IL-8 promoter. The constitutive and cytokine IL-1-induced promoter activity of the chemokine MGSA/GRO alpha in normal retinal pigment epithelial and the Hs294T melanoma cells is partially regulated through the NF-kappa B element, which binds both NF-kappa B p50 and RelA (NF-kappa B p65) homodimers and heterodimers. Mutational analysis of the MGSA/GRO alpha promoter reveals that, in addition to the NF-kappa B element, the immediate upstream region (IUR) is necessary for basal expression in retinal pigment epithelial and Hs294T cells. Gel mobility shift and UV cross-linking analyses demonstrate that several constitutive DNA binding proteins interact with the IUR. Although this region has sequence similarity to the several transcription factor elements including C/EBP, the IUR includes sequences that have no similarity to previously identified enhancer regions. Furthermore, RelA transactivates through either the NF-kappa B element or the IUR, suggesting a putative interaction between NF-kappa B and this novel complex.