We aimed to investigate various anti-neuronal antibodies in sera of amyotrophic lateral sclerosis (ALS) patients to detect possible autoimmune encephalitis patients imitating ALS findings and to delineate the validity of routine screening of well-characterized anti-neuronal antibodies in ALS. The patients fulfilling the revised El Escorial diagnostic criteria for definite ALS were included. Their serum samples were investigated for antiganglioside (IgM/IgG) and onconeural (IgG) antibodies by immunoblotting, for ion channel antibodies (IgG) by a cell-based assay and for IgG binding patterns to the rat brain by indirect immunohistochemistry. Thirty-five patients with definite ALS and 30 healthy individuals were included. Ganglioside antibodies were detected in 2 out of 35 (5.7%) patients with ALS. The onconeural and ion channel antibodies were negative in our series. Varied serum IgG binding patterns were identified in eight (22.9%) patients. Although autoimmune encephalitis patients may occasionally present with atypical motor neuron disease findings, definite ALS patients do not appear to exhibit onconeural or ion channel antibodies, suggesting that routine analysis of these antibodies in typical ALS is not mandatory. By contrast, some ALS patients display anti-neuronal antibodies against undetermined target antigens, prompting investigation of these novel antibodies with more advanced methods.