Permanent human small cell lung cancer (SCLC) cell lines established in our laboratory were investigated for their expression of the enzymatic neuroendocrine markers L-DOPA decarboxylase (DDC), neuron-specific enolase (NSE), and creatine kinase (CK), including its BB isoenzyme (CK-BB), the classical tumor markers carcinoembryonic antigen (CEA), the alpha and beta subunits of human chorionic gonadotropin (alpha-HCG, beta-HCG), and alpha-fetoprotein (alpha-FP), and their chromosomal characteristics. DDC activities were detectable in 5/6 SCLC cell lines and absent in non-SCLC. NSE levels ranged from 160 to 1422 ng/mg soluble protein and were less than 290 ng/mg soluble protein in non-SCLC. Activities of CK and levels of CK-BB clearly distinguished SCLC from non-SCLC with CK activities greater than 1000 munits/mg soluble protein and CK-BB levels greater than 3000 ng/mg soluble protein in SCLC and less than 300 munits/mg soluble protein and less than 2000 ng/mg soluble protein in non-SCLC. CEA was detectable in 5/6 SCLC cell lines but absent in non-SCLC, and its level seemed to correlate with those of DDC, NSE, and CK. One cell line, SCLC-16H, lost some of its neuroendocrine properties and CEA after 1 year of in vitro cultivation. Generally, marker levels were low in fast growing cell lines and high in slow growing cell lines. HCG alpha and beta subunit and alpha-FP were not detectable in SCLC cell lines. All SCLC cell lines examined had near diploid DNA indices and modal chromosome numbers. Double minute chromosomes and homogeneously staining regions were found in 2/5 and 4/5 SCLC cell lines respectively. With respect to chromosomal aberrations, we found a deletion of the short arm of at least one chromosome 3 in all SCLC cell lines (5/5). These data show that SCLC expresses neuroendocrine markers and CEA; CK is the most sensitive marker, and DDC and CEA are the most specific markers for SCLC in vitro; individual marker levels correlate with each other and the in vitro malignancy of SCLC; and SCLC cell lines have relatively uniform chromosomal characteristics. Our results suggest that patients whose tumors have high levels of DDC, NSE, CK-BB, and CEA have a better prognosis than those with low marker levels. This hypothesis could be proved by comparing pairs of patients that are matched for all known prognostic parameters, in particular tumor spread, for their serum and tumor marker levels with respect to the patients' outcome and prognosis.