The δ13C of pine needle CO2 evolved in darkness (δ13Cr) for slash pine trees (Pinus elliottii) was determined by placing recently collected pine needles in darkness and collecting respired CO2 over a short time period (< 15 min). δ13Cr measurements were made over several 24 h periods to test the hypothesis that significant variation in δ13Cr would be observed during a diurnal cycle. The δ13Cr measurements from the 24 h time series trials showed a consistent midday 13C-enrichment (5–10‰) relative to bulk biomass. The δ13Cr values became more 13C-depleted at night and following shading, and approached bulk-biomass δ13C values by dawn. The effect of night-time respired 13C-enriched CO2 on the δ13C value of the remaining assimilate is shown to be minimal (13C depleted by 0.22‰) under field conditions for P. elliottii needles.