Cytohesin-1 and ARNO are guanine nucleotide-exchange factors (GEFs) for ADP-ribosylation factor (Arf). Here, we show that ARNO is expressed in HL-60 cells and established that granulocytic differentiation induced with Me2SO stimulated cytohesin-1 but not ARNO expression. Cytohesin-1 levels in HL-60 granulocytes were similar to those in human neutrophils. Me2SO-differentiated HL-60 cells expressed ARNO and cytohesin-1 isoforms with a diglycine and a triglycine motif in their PH domains, respectively. In vitro, ARNO diglycine and cytohesin-1 triglycine enhanced phospholipase D1 (PLD1) activation by Arf1 with near-maximal effects at 250 nM. These effects were marked particularly at low Mg2+ concentrations. PLD activation was well-correlated with GTP binding to Arf1, and cytohesin-1 was always more potent than ARNO in the PLD- and GTP-binding assays. Increasing Mg2+ concentrations reduced PLD and Arf1 activation by Arf-GEFs. fMetLeuPhe and phorbol 12-myristate 13-acetate stimulated ARNO and cytohesin-1 as well as Arf1 translocation to HL-60 cell membranes. fMetLeuPhe-mediated ARNO recruitment, but not cytohesin-1 and Arf1 translocation, was blocked by phosphatidylinositol 3-kinase inhibitors. The combined results demonstrate that cytohesin-1 triglycine participates in a major phosphatidylinositol 3-kinase-independent pathway linking cell-surface receptors to Arf1 activation and translocation in human granulocytes.