Studies have shown that He–Ne laser irradiation can affect the biological functions of macrophages. The aim of the present study was to investigate the effect of He–Ne laser irradiation on the various functional parameters of macrophages and look for possible correlations in the effects to understand the mechanisms involved. Mice peritoneal macrophages were irradiated with a He–Ne laser (632.8 nm, ∼10 W m−2) at energy densities ranging from 100 to 600 J m−2 and the activities of lysozyme and cathepsin, phagocytosis, and cell spreading (markers of cell activation), as well as changes in NAD(P)H autofluorescence, were monitored. He–Ne laser irradiation was observed to lead to significant changes in all the parameters investigated. While lysozyme activity and spreading of the peripheral membrane were found to increase with the irradiation dose over the dose range investigated, the phagocytotic activity of macrophages, the activity of cathepsin, the observed decease in cell membrane fluidity and the observed increase in NAD(P)H level showed a peak at 200 J m−2. Possible reasons for and the significance of the observed correlations are discussed.