The zebrafish is an outstanding model for intravital imaging of inflammation due to its optical clarity and the ability to express fluorescently labelled specific cell types by transgenesis. However, although several transgenic labelling myeloid cells exist, none allow distinction of macrophages from neutrophils. This prevents simultaneous imaging and examination of the individual contributions of these important leukocyte subtypes during inflammation. We therefore used Bacterial Artificial Chromosome (BAC) recombineering to generate a transgenic Tg(fms:GAL4.VP16)i186, in which expression of the hybrid transcription factor Gal4-VP16 is driven by the fms (CSF1R) promoter. This was then crossed to a second transgenic expressing a mCherry-nitroreductase fusion protein under the control of the Gal4 binding site (the UAS promoter), allowing intravital imaging of mCherry-labelled macrophages. Further crossing this compound transgenic with the neutrophil transgenic Tg(mpx:GFP)i114 allowed clear distinction between macrophages and neutrophils and simultaneous imaging of their recruitment and behaviour during inflammation. Compared with neutrophils, macrophages migrate significantly more slowly to an inflammatory stimulus. Neutrophil number at a site of tissue injury peaked around 6 hours post injury before resolving, while macrophage recruitment increased until at least 48 hours. We show that macrophages were effectively ablated by addition of the prodrug metronidazole, with no effect on neutrophil number. Crossing with Tg(Fli1:GFP)y1 transgenic fish enabled intravital imaging of macrophage interaction with endothelium for the first time, revealing that endothelial contact is associated with faster macrophage migration. Tg(fms:GAL4.VP16)i186 thus provides a powerful tool for intravital imaging and functional manipulation of macrophage behaviour during inflammation.