Photosynthesis is a complex process whose rate is affected by many biochemical and biophysical factors. Fortunately, it is possible to determine, or at least estimate, many of the most important parameters using a combination of optical methods and gas transient analyses. We describe here a computer-operated routine that has been developed to make detailed assessments of photosynthesis at a comprehensive level. The routine comprised the following measurements: steady-state light and CO2 response curves of net CO2 assimilation at 21 and 2 kPa O2; transients from limiting to different saturating CO2 concentrations at 2 kPa O2; post-illumination CO2 fixation transient; dark–light induction of O2 evolution; O2 yield from one saturating single-turnover flash; chlorophyll fluorescence F0, Fs and Fm during the light and CO2 response curves; leaf transmission at 820 nm (P700+) during the light and CO2 response curves; post-illumination re-reduction time of P700+. The routine was executed on a two-channel fast-response gas exchange measurement system (A. Laisk and V. Oja: Dynamic Gas Exchange of Leaf Photosynthesis. CSIRO, Canberra, Australia). Thirty-six intrinsic characteristics of the photosynthetic machinery were derived, including quantum yield of CO2 fixation (YCO2), time constant of P700 re-reduction (τ′), relative optical cross-sections of PSII and PSI antennae (aII, aI), PSII and PSI density per leaf area unit, plastoquinone pool, total mesophyll resistance, mesophyll diffusion resistance, Vm, Km(CO2) and CO2/O2 specificity of Rubisco, RuBP pool at CO2 limitation (assimilatory charge). An example of the routine and calculations are shown for one leaf and data are presented for leaves of 8-year-old-trees of two birch clones growing in Suonenjoki Forest Research Station, Finland, during summer 2000. Parameters YCO2, basic τ′, aII, aI, Km(CO2) and Ks varied little in different leaves [relative standard deviation (RSD) < 7%], other parameters scattered widely (RSD typically 10–40%). It is concluded that the little scattered parameters are determined by basic physico-chemical properties of the photosynthetic machinery whereas the widely scattered parameters are adjusting to growth conditions. The proposed non-destructive routine is suitable for diagnosing the photosynthetic machinery of leaves and may be applied in plant ecophysiology and in genetic engineering of plants.