OBJECTIVE To investigate the interaction between hydrogen sulfide (H2S)/cystathionine gamma-lyase (CSE) and carbon monoxide (CO)/heme oxygenase (HO) pathways in aortic smooth muscle cells (ASMC). METHODS The ASMCs were divided into the following groups: (1) the control group; (2) the zinc protoporphyrin (ZnPP) 20 micromol/L group; (3) the propargylglycine (PPG) 2 mmol/L, 4 mmol/L and 10 mmol/L groups; and (4) the sodium hydrosulfide (NaHS) 1 x 10(-5) mol/L, 1 x 10(-4) mol/L and 1 x 10(-3) mol/L groups. Each of the groups was further divided into 6 h, 12 h, 18 h and 24 h subgroups. The CO level, represented by carboxyhemoglobin (HbCO) content was measured using a spectrophotometric method and H2S content was detected by a sensitive electrode method. CSE and HO-1 expressions were detected by Western blotting. RESULTS The H2S content in the medium and CSE expression by ASMC were markedly increased by ZnPP compared with the control group. HbCO content in the medium and HO-1 expression by the ASMC started strengthening following 24 h treatment with PPG at 2 mmol/L, but were further strengthened following 18 h and 24 h treatment with PPG at 4 mmol/L compared with the controls (P < 0.01). PPG at 10 mmol/L increased the HbCO level in the medium following 18 h treatment and increased HO-1 expression by the ASMC following 12 h treatment. Moreover, NaHS at 1 x 10(-5) mol/L and 1 x 10(-4) mol/L decreased the HbCO level in the medium and HO-1 expression by the ASMC after 6 h and 12 h treatment, while NaHS at 1 x 10(-3) mol/L decreased them at all time points of the treatments. CONCLUSIONS The results suggested that endogenous CO/HO and H2S/CSE pathways inhibited each other in ASMC under physiological conditions.