We have investigated several factors which influence acetyl-CoA carboxylase (ACCase) activity in lysed spinach chloroplasts. (1) When assayed after rapid lysis of light-incubated chloroplasts, ACCase activity was 2-fold higher than activity from dark-incubated chloroplasts. Within 5 min after lysis, activity from dark-incubated chloroplasts increased, suggesting a transient inactivation or inhibition of ACCase in the dark. (2) When lysed chloroplast suspensions were incubated with 30 to 100 microM acetyl-CoA before starting assays, activity was 4-fold higher than if suspensions were not preincubated with acetyl-CoA. CoA, malonyl-CoA, propionyl-CoA, and butyryl-CoA also activated ACCase. Full acetyl-CoA activation required MgATP and was essentially complete after 8 min. ACCase activity decreased upon removal of acetyl-CoA by gel filtration and was partially restored by readdition of acetyl-CoA. Thus, ACCase activation by acetyl-CoA was reversible. (3) Dithiothreitol and thioredoxin stimulated ACCase activity, but only in preparations where ACCase activity was low. (4) ACCase was assayed in concentrations of ATP, ADP, NADPH, NADP+, Mg2+, and CO2/HCO-3, which are estimated to occur in the stroma of chloroplasts under illumination or darkness. ACCase activity from lysed chloroplast suspensions was 10-fold higher when illuminated conditions were used. However, this activity was still 5-fold to 10-fold lower than the rates required to sustain known in vivo rates of fatty acid synthesis and in vitro rates achieved under optimum assay conditions with saturating substrates.