Periphyton distribution is affected by various physical, chemical and biological factors at different scales. In the present study, a cheap and rapid method of measurement was used to obtain the many samples necessary for spatial analysis of periphyton along an altitudinal gradient in a low-order stream in south-east Brazilian Atlantic rain-forest. Fluorescence and turbidity were measured using a hand-held fluorometer and calibrated to the chlorophyll and dry mass of periphyton. Periphyton on the horizontal and vertical surfaces of different boulders was examined and shading and water current were measured as covariables. The three upstream sites with higher abundances of potentially grazing and bioturbing shrimps and mayflies had significantly less periphyton dry mass than the three downstream sites. Chlorophyll was positively related to water current, but not to shading. Variabilities in the dry mass and chlorophyll among boulders within sites were not associated with the distribution of shrimps and mayflies. The in vivo measurement was cheap, rapid, sensitive and reasonably precise compared with standard methods. The necessary sacrifice of detail of pigments (different chlorophylls and pheophytin) and dry mass (organic and inorganic constituents) and probably precision was compensated for by the insights gained from the ability to obtain a large number of samples in a hierarchical design.